Protein-tyrosine kinase genes

ABSTRACT

Substantially pure receptor PTK subtypes and methods of using the subtypes are provided.

CROSS-RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.No. 08/456,647, filed Jun. 2, 1995, issued on Sep. 22, 1998 as U.S. Pat.No. 5,811,516, which is a divisional of U.S. patent application Ser.No.08/237,401, filed May 2, 1994, which is a continuation of U.S. patentapplication Ser. No. 07/884,486, filed May 15, 1992.

[0002] This work was supported by Grant Number NS-23896 from theNational Institutes of Health. The United States Government may retaincertain rights of this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates generally to the molecular cloning ofgenes which encode unique protein-tyrosine kinase receptor subtypeswhich can be used in an assay to screen various compositions whichmodulate these receptors.

[0005] 2. Related Art

[0006] Among the signal transduction molecules implicated in neuraldevelopment, the receptor protein-tyrosine kinases (PTKs) are ofparticular interest: These proteins function as transmembrane receptorsfor polypeptide growth factors, and contain a tyrosine kinase as anintegral part of their cytoplasmic domains (Yarden and Ullrich, Annu.Rev. Biochem., 57:443-478, 1988; Ullrich and Schlessinger, Cell,61:203-212, 1990). Binding of a polypeptide ligand to its correspondingcell surface receptor results in rapid activation of that receptor'sintracellular tyrosine kinase, which in turn results in the tyrosinephosphorylation of the receptor itself and of multiple downstream targetproteins (Hunter and Cooper, Annu. Rev. Biochem., 54:897-930, 1985;Hunter, et al, eds. J. B. Hook and G. Poste, Plenum Press, New York andLondon, pp. 119-139, 1990). For many receptor PTKs, growth factorbinding ultimately triggers multiple rounds of cell division.

[0007] Molecular studies of mutations that affect cell differentiationhave demonstrated that several of these receptor PTKs act as earlydeterminants of cell fate. Loss-of-function mutations in the sevenlessgene of Drosophila (Harris, et al., J. Physiol., 256:415-439, 1976), forexample, abolish the tyrosine kinase activity of a transmembranereceptor expressed in the developing ommatidia of the eye and result inthe aberrant differentiation of the precursors to the number 7photoreceptors (Basler and Haten, Cell, 54:299-311, 1988; Rubin, Cell,57:519-520, 1989). Rather than becoming number 7 photoreceptor cells,these precursors instead differentiate into non-neuronal cone cells,which form the lens. In marked contrast, the remaining complement ofphotoreceptors (numbers 1-6 and 8) differentiate normally.

[0008] Mutations in genes encoding other receptor PTKs have also beenshown to affect cell differentiation. For example, mutations in thetorso gene of Drosophila specifically disrupt the terminaldifferentiation of extreme anterior and posterior structures in theembryo (Sprenger, et al., Nature 338:478-483, 1989), and mutations inthe Drosophila Ellipse gene, which encodes a homolog of the mammalianepidermal growth factor (EGF) receptor, result in the developmentalfailure of multiple cell types in the eye (Baker and Rubin, Nature,340:150-153, 1989). In vertebrates, mutations in the mouse dominantwhite spotting locus (W), which encodes the c-kit receptor PTK, producepleiotropic developmental effects that include disruption of the normalproliferation and differentiation of neural crest-derived melanocytes(Chabot, et al., Nature, 335:88-89, 1988; Geissler, et al., Cell,55:185-192, 1988).

[0009] Parallel to these studies of the developmental role of receptorPTKs has been the demonstration that many of the ligands for thesereceptors influence the differentiation of neural cells in culture.Platelet-derived growth factor (PDGF), for example, has been shown tostimulate the proliferation and prevent the premature differentiation ofoligodendrocyte/type-2 astrocyte glial progenitor cells in rat opticnerve cultures (Noble, et al., Nature, 333:560-562, 1988; Raff, et al.,Nature, 333:562-565, 1988).

[0010] Similarly, both acidic and basic fibroblast growth factor (bFGF)have been shown to stimulate the neuronal differentiation of culturedrat pheochromocytoma (PC-12) cells (Togari, et al., J. Neurosci.,5:307-316, 1985; Wagner and D'Amore, J. Cell Biol., 103:1363-1367,1986). bFGF has also been reported to prolong survival and stimulateneurite outgrowth in cultures of primary cortical and hippocampalneurons (Morrison, et al., Proc. Natl. Acad. Sci. USA, 83:7537-7541,1986; Walicke, et al., Proc. Natl. Acad. Sci. USA, 83:3012-3016, 1986),to induce cell division, neuronal differentiation, and nerve growthfactor (NGF) dependence in adrenal chromatifin cells (Stemple, et al.,Neuron, 1:517-525, 1988), and to function as a survival factor, both invivo and in vitro, for neural crest-derived non-neuronal cells duringthe early development of sensory ganglia (Kalcheim, Dev. Biol.,134:1-10, 1989). Recently, the product of the mouse mutant steel gene(Sl), which interacts genetically with W, has been identified as agrowth factor ligand for the c-kit receptor (Witte, Cell, 63:5-6, 1990).Genetic and biochemical studies of the expression patterns of thesevenless, torso and c-kit receptors suggest that specification of cellfates can be achieved through the spatially and temporally restrictedexpression of either the receptors or their ligands (Rubin, Cell,57:519-520, 1989; Tomlinson and Ready, Biol., 120:366-376, 1987; Reinkeand Zipursky, Cell, 55:321-330, 1988; Banerjee and Zipursky, Neuron,4:177-187, 1990; Stevens, et al., Nature, 346:660-663, 1990; Matsui, etal., Nature, 347:667-669, 1990).

SUMMARY OF THE INVENTION

[0011] In accordance with the present invention, novel receptor proteintyrosine kinase (PTK) subtype polypeptides have been isolated. ThesePTKs possess a tyrosine kinase domain and a unique tissue expressionpattern different from all previously known receptor PTKs. These novelreceptor PTK subtypes have been designated tyro-1 through tyro-8 andtyro-10 through tyro-12. Of particular interest among the new PTKsubtypes are tyro-1 through tyro-6 which are found predominantly orexclusively in neural tissue.

[0012] By providing the polynucleotide sequences and correspondingpolypeptide sequences for the new PTK subtypes, it is now possible toobtain polynucleotide sequences encoding the entire receptor PTK foreach of the subtypes.

[0013] Further, the invention provides a method for identifyingcompositions which potentially affect the activity of the receptor PTKsubtype. This method comprises (a) contacting cells containing DNA whichexpresses the PTK polypeptide with the composition under conditionssuitable for cell culture; and (b) monitoring the cells for aphysiological change resulting from this interaction.

[0014] In addition, the present invention provides uniqueoligonucleotide which align with the unique flanking regions of thereceptor PTK subtypes, thereby allowing amplification of thepolynucleotides encoding the receptor PTK subtype by such techniques aspolymerase chain reaction (PCR).

[0015] The present invention also provides a method of gene therapycomprising introducing into a host subject an expression vectorcomprising a nucleotide sequence encoding a receptor PTK subtype capableof affecting a biological activity of the host subject cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIGS. 1A and 1B show the tissue expression profiles of the novelPTK mRNAs.

[0017]FIG. 2 shows the developmental tissue profiles of the novel PTKmRNAs which were predominantly or exclusively neural in theirdistribution.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0018] The present invention relates to novel protein tyrosine kinase(PTK) gene and polypeptides encoded by these genes. Various of these PTKsubtypes are implicated in neural development where they functionprimarily as signal transduction molecules. The receptor PTKs of theinvention are characterized as having a tyrosine kinase domain and aunique tissue expression pattern which differs from that of all knownreceptor PTKs.

[0019] The invention provides polynucleotides, such as DNA, cDNA, andRNA, encoding novel receptor PTK polypeptides. It is understood that allpolynucleotides encoding all or a portion of the receptor PTKs of theinvention are also included herein, so long as they exhibit at least oneprotein tyrosine kinase domain and the tissue expression patterncharacteristic of a given subtype. Such polynucleotides include bothnaturally occurring and intentionally manipulated, for example,mutagenized polynucleotides.

[0020] DNA sequences of the invention can be obtained by severalmethods. For example, the DNA can be isolated using hybridizationprocedures which are well known in the art. These include, but are notlimited to: 1) hybridization of probes to genomic or cDNA libraries todetect shared nucleotide sequences and 2) antibody screening ofexpression libraries to detect shared structural features.

[0021] Hybridization procedures are useful for the screening ofrecombinant clones by using labeled mixed synthetic oligonucleotideprobes where each probe is potentially the complete complement of aspecific DNA sequence in the hybridization sample which includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. In other words, by using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific cDNA clone by the hybridization of the target DNA to thatsingle probe in the mixture which is its complete complement (Wallace,et al., Nucleic Acid Research, 9:879, 1981).

[0022] A receptor PTK cDNA library can be screened by injecting thevarious cDNAs into oocytes, allowing sufficient time for expression ofthe cDNA gene products to occur, and testing for the presence of thedesired cDNA expression product, for example, by using antibody specificfor the receptor PTK subtype polypeptide or by using functional assaysfor receptor PTK subtype activity and a tissue expression patterncharacteristic of the desired subtype.

[0023] Alternatively, a cDNA library can be screened indirectly forreceptor PTK polypeptides having at least one epitope using antibodiesspecific for receptor PTK subtypes of the invention. Such antibodies canbe either polyclonally or monoclonally derived and used to detectexpression product indicative of the presence of protein tyrosine kinasereceptor PTK subtype cDNA.

[0024] The development of specific DNA sequences encoding receptor PTKsubtypes of the invention can also be obtained by: (1) isolation of adouble-stranded DNA sequence from the genomic DNA; (2) chemicalmanufacture of a DNA sequence to provide the necessary codons for thepolypeptide of interest; and (3) in vitro synthesis of a double-strandedDNA sequence by reverse transcription of mRNA isolated from a eukaryoticdonor cell. In the latter case, a double-stranded DNA complement of mRNAis eventually formed which is generally referred to as cDNA.Specifically embraced in (1) are genomic DNA sequences which encodeallelic variant forms. Also included are DNA sequences which aredegenerate as a result of the genetic code.

[0025] Of the three above-noted methods for developing specific DNAsequences for use in recombinant procedures, the use of genomic DNAisolates (1), is the least common. This is especially true when it isdesirable to obtain the microbial expression of mammalian polypeptidesbecause of the presence of introns.

[0026] The synthesis of DNA sequences is frequently the method of choicewhen the entire sequence of amino acid residues of the desiredpolypeptide product is known. When the entire sequence of amino acidresidues of the desired polypeptide is not known, the direct synthesisof DNA sequences is not possible and the method of choice is theformation of cDNA sequences. Among the standard procedures for isolatingcDNA sequences of interest is the formation of plasmid-carrying cDNAlibraries which are derived from reverse transcription of mRNA which isabundant in donor cells that have a high level of genetic expression.When used in combination with polymerase chain reaction technology, evenrare expression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucleic Acid Research, 11:2325,1983).

[0027] Since the novel DNA sequences of the invention encode essentiallyall or part of a receptor PTK, it is now a routine matter to prepare,subclone, and express smaller polypeptide fragments of DNA from this orcorresponding DNA sequences. Alternatively, by utilizing the DNAfragments disclosed herein which define the unique tyrosine kinasereceptor subtype of the invention it is possible, in conjunction withknown techniques, to determine the DNA sequences encoding the entirereceptor subtypes. Such techniques are described in U.S. Pat. Nos.4,394,443 and 4,446,235 which are incorporated herein by reference.

[0028] The polypeptide resulting from expression of a DNA sequence ofthe invention can be further characterized as being free fromassociation with other eukaryotic polypeptides or other contaminantswhich might otherwise be associated with the protein kinase in itsnatural cellular environment.

[0029] Isolation and purification of microbially expressed polypeptidesprovided by the invention may be by conventional means including,preparative chromatographic separations and immunological separationsinvolving monoclonal and/or polyclonal antibody preparation.

[0030] For purposes of the present invention, receptor PTK subtypeswhich are homologous to those of the invention can be identified bystructural as well as functional similarity. Structural similarity canbe determined, for example, by assessing polynucleotide strandhybridization or by screening with antibody, especially a monoclonalantibody, which recognizes a unique epitope present on the subtypes ofthe invention. When hybridization is used as criteria to establishstructural similarity, those polynucleotide sequences which hybridizeunder stringent conditions to the polynucleotides of the invention areconsidered to be essentially the same as the polynucleotide sequences ofthe invention.

[0031] In the present invention, polynucleotide sequences encodingreceptor PTK subtype may be introduced into a host cell by means of arecombinant expression vector. The term “recombinant expression vector”refers to a plasmid, virus or other vehicle known in the art that hasbeen manipulated by insertion or incorporation of the polynucleotidesequences of the invention. Such expression vectors typically contain apromotor sequence which facilitates efficient transcription of theinserted sequence in the host. The expression vector also typicallycontains specific genes which allow phenotypic selection of thetransformed cells. Alternatively, nucleotide sequences encoding areceptor PTK subtype can be introduced directly in the form of freenucleotide, for example, by microinjection, or transfection.

[0032] DNA sequences encoding receptor PTK subtypes of the invention canbe expressed in vivo by DNA transfer into a suitable host cell.“Recombinant host cells” or “host cells” are cells in which a vector canbe propagated and its DNA expressed. The term includes not onlyprokaryotes, but also such eukaryotes as yeast, filamentous fungi, aswell as animal cells which can replicate and express an intron-free DNAsequence of the invention and any progeny of the subject host cell. Itis understood that not all progeny are identical to the parental cellsince there may be mutations that occur at replication. However, suchprogeny are included when the terms above are used.

[0033] Methods of expressing DNA sequences having eukaryotic codingsequences in prokaryotes are well known in the art. Biologicallyfunctional viral and plasmid DNA vectors capable of expression andreplication in a host are known in the art. Such vectors are used toincorporate DNA sequences of the invention. Hosts include microbial,yeast and mammalian organisms.

[0034] Transformation of a host cell with recombinant DNA may be carriedout by conventional techniques which are well known to those skilled inthe art. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method well known in the art. Alternatively, MgCl₂ or RbCl can be used.Transformation can also be performed after forming a protoplast of thehost cell.

[0035] When the host is a eukaryote, such methods of transfection of DNAas calcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, insertion of a plasmid encased in liposomes, orvirus vectors may be used. Eukaryotic cells can also be cotransformedwith foreign cDNA encoding the desired receptor PTK subtype protein, anda second foreign DNA molecule encoding a selectable phenotype, such asthe herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982).

[0036] Where the eukaryotic host cells are yeast, the cDNA can beexpressed by inserting the cDNA into appropriate expression vectors andintroducing the product into the host cells. Various shuttle vectors forthe expression of foreign genes in yeast have been reported (Heinemann,et al., Nature, 340:205, 1989; Rose, et al., Gene, 60:237, 1987).

[0037] Isolation and purification of microbially expressed protein, orfragments thereof provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.Antibodies provided in the present invention are immunoreactive with thereceptor PTK subtypes of the invention. Antibody which consistsessentially of pooled monoclonal antibodies with different epitopicspecificities, as well as distinct monoclonal antibody preparations areprovided. Monoclonal antibodies are made from antigen containingfragments of the protein by methods well known in the art (Kohler, etal., Nature, 256:495, 1975; Current Protocols in Molecular Biology,Ausubel, et al., ed., 1989).

[0038] Minor modifications of the receptor PTK primary amino acidsequence may result in proteins which have substantially equivalentactivity compared to the receptor PTKs described herein. Suchmodifications may be deliberate, as by site-directed mutagenesis, or maybe spontaneous. All proteins produced by these modifications areincluded herein as long as tyrosine kinase activity and thecharacteristic tissue expression pattern for the subtype is present.

[0039] The invention also discloses a method for identifying acomposition which affects the activity of a receptor PTK subtype of theinvention. The receptor is, for example, capable of affecting celldivision and/or differentiation. The composition is incubated incombination with cells under conditions suitable for cell culture, thensubsequently monitoring the cells for a physiologic change.

[0040] The production of a receptor PTK can be accomplished byoligonucleotide(s) which are primers for amplification of the genomicpolynucleotide encoding PTK receptor. These unique oligonucleotideprimers were produced based upon identification of the flanking regionscontiguous with the polynucleotide encoding the receptor PTK. Theseoligonucleotide primers comprise sequences which are capable ofhybridizing with the flanking nucleotide sequence encoding a polypeptidehaving amino acid residues HRDLAAR and/or DVWS(F/Y)G(V/I) and sequencescomplementary thereto.

[0041] The primers of the invention embrace oligonucleotides ofsufficient length and appropriate sequence so as to provide specificinitiation of polymerization on a significant number of nucleic acids inthe polynucleotide encoding the receptor PTK subtype. Specifically, theterm “primer” as used herein refers to a sequence comprising two or moredeoxyribonucleotides or ribonucleotides, preferably more than three,which sequence is capable of initiating synthesis of a primer extensionproduct, which is substantially complementary to a receptor PTK strand.Environmental conditions conducive to synthesis include the presence ofnucleoside triphosphates and an agent for polymerization, such as DNApolymerase, and a suitable temperature and pH. The primer is preferablysingle stranded for maximum efficiency in amplification, but may bedouble stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent for polymerization. The exact length ofprimer will depend on many factors, including temperature, buffer, andnucleotide composition. The oligonucleotide primer typically contains15-22 or more nucleotides, although it may contain fewer nucleotides.

[0042] Primers of the invention are designed to be “substantially”complementary to each strand of polynucleotide encoding the receptor PTKto be amplified. This means that the primers must be sufficientlycomplementary to hybridize with their respective strands underconditions which allow the agent for polymerization to perform. In otherwords, the primers should have sufficient complementarity with theflanking sequences to hybridize therewith and permit amplification ofthe polynucleotide encoding the receptor PTK. Preferably, the primershave exact complementarity with the flanking sequence strand.

[0043] Oligonucleotide primers of the invention are employed in theamplification process which is an enzymatic chain reaction that producesexponential quantities of polynucleotide encoding the receptor PTKrelative to the number of reaction steps involved. Typically, one primeris complementary to the negative (−) strand of the polynucleotideencoding the receptor PTK and the other is complementary to the positive(+) strand. Annealing the primers to denatured nucleic acid followed byextension with an enzyme, such as the large fragment of DNA Polymerase I(Klenow) and nucleotides, results in newly synthesized + and −strandscontaining the receptor PTK sequence. Because these newly synthesizedsequences are also templates, repeated cycles of denaturing, primerannealing, and extension results in exponential production of the region(i.e., the tyrosine kinase receptor polynucleotide sequence) defined bythe primer. The product of the chain reaction is a discrete nucleic acidduplex with termini corresponding to the ends of the specific primersemployed.

[0044] The oligonucleotide primers of the invention may be preparedusing any suitable method, such as conventional phosphotriester andphosphodiester methods or automated embodiments thereof. In one suchautomated embodiment, diethylphos-phoramidites are used as startingmaterials and may be synthesized as described by Beaucage, et al.(Tetrahedron Letters, 22:1859-1862, 1981). One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066.

[0045] Any nucleic acid specimen, in purified or nonpurified form, canbe utilized as the starting nucleic acid or acids, provided it contains,or is suspected of containing, the specific nucleic acid sequencecontaining a protein receptor PTK of the invention. Thus, the processmay employ, for example, DNA or RNA, including messenger RNA, whereinDNA or RNA may be single stranded or double stranded. In the event thatRNA is to be used as a template, enzymes, and/or conditions optimal forreverse transcribing the template to DNA would be utilized. In addition,a DNA-RNA hybrid which contains one strand of each may be utilized. Amixture of nucleic acids may also be employed, or the nucleic acidsproduced in a previous amplification reaction herein, using the same ordifferent primers may be so utilized. The specific nucleic acid sequenceto be amplified, i.e., the receptor PTK, may be a fraction of a largermolecule or can be present initially as a discrete molecule, so that thespecific sequence constitutes the entire nucleic acid. It is notnecessary that the sequence to be amplified be present initially in apure form; it may be a minor fraction of a complex mixture, such ascontained in whole human DNA.

[0046] DNA or RNA utilized herein may be extracted from a body sample,such as brain, or various other tissue, by a variety of techniques suchas that described by Maniatis, et al. (Molecular Cloning, 280:281,1982). If the extracted sample is impure (such as plasma, serum, orblood), it may be treated before amplification with an amount of areagent effective to open the cells, fluids, tissues, or animal cellmembranes of the sample, and to expose and/or separate the strand(s) ofthe nucleic acid(s). This lysing and nucleic acid denaturing step toexpose and separate the strands will allow amplification to occur muchmore readily.

[0047] Where the target nucleic acid sequence of the sample contains twostrands, it is necessary to separate the strands of the nucleic acidbefore it can be used as the template. Strand separation can be effectedeither as a separate step or simultaneously with the synthesis of theprimer extension products. This strand separation can be accomplishedusing various suitable denaturing conditions, including physical,chemical, or enzymatic means, the word “denaturing” includes all suchmeans. One physical method of separating nucleic acid strands involvesheating the nucleic acid until it is denatured. Typical heatdenaturation may involve temperatures ranging from about 80° to 105° C.for times ranging from about 1 to 10 minutes. Strand separation may alsobe induced by an enzyme from the class of enzymes known as helicases orby the enzyme RecA, which has helicase activity, and in the presence ofriboATP, is known to denature DNA. The reaction conditions suitable forstrand separation of nucleic acids. with helicases are described by KuhnHoffmann-Berling (CSH-Quantitative Biology, 43:63, 1978) and techniquesfor using RecA are reviewed in C. Radding (Ann. Rev. Genetics,16:405-437, 1982).

[0048] If the nucleic acid containing the sequence to be amplified issingle stranded, its complement is synthesized by adding one or twooligonucleotide primers. If a single primer is utilized, a primerextension product is synthesized in the presence of primer, an agent forpolymerization, and the four nucleoside triphosphates described below.The product will be partially complementary to the single-strandednucleic acid and will hybridize with a single-stranded nucleic acid toform a duplex of unequal length strands that may then be separated intosingle strands to produce two single separated complementary strands.Alternatively, two primers may be added to the single-stranded nucleicacid and the reaction carried out as described.

[0049] When complementary strands of nucleic acid or acids areseparated, regardless of whether the nucleic acid was originally doubleor single stranded, the separated strands are ready to be used as atemplate for the synthesis of additional nucleic acid strands. Thissynthesis is performed under conditions allowing hybridization ofprimers to templates to occur. Generally synthesis occurs in a bufferedaqueous solution, preferably at a pH of 7-9, most preferably about 8.Preferably, a molar excess (for genomic nucleic acid, usually about10⁸:1 primer:template) of the two oligonucleotide primers is added tothe buffer containing the separated template strands. It is understood,however, that the amount of complementary strand may not be known if theprocess of the invention is used for diagnostic applications, so thatthe amount of primer relative to the amount of complementary strandcannot be determined with certainty. As a practical matter, however, theamount of primer added will generally be in molar excess over the amountof complementary strand (template) when the sequence to be amplified iscontained in a mixture of complicated long-chain nucleic acid strands. Alarge molar excess is preferred to improve the efficiency of theprocess.

[0050] The deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTPare added to the synthesis mixture, either separately or together withthe primers, in adequate amounts and the resulting solution is heated toabout 90°-100° C. from about 1 to 10 minutes, preferably from 1 to 4minutes. After this heating period, the solution is allowed to cool toroom temperature, which is preferable for the primer hybridization. Tothe cooled mixture is added an appropriate agent for effecting theprimer extension reaction (called herein “agent for polymerization”),and the reaction is allowed to occur under conditions known in the art.The agent for polymerization may also be added together with the otherreagents if it is heat stable. This synthesis (or amplification)reaction may occur at room temperature up to a temperature above whichthe agent for polymerization no longer functions.

[0051] Thus, for example, if DNA polymerase is used as the agent, thetemperature is generally no greater than about 40 ° C. Most convenientlythe reaction occurs at room temperature.

[0052] The agent for polymerization may be any compound or system whichwill function to accomplish the synthesis of primer extension products,including enzymes. Suitable enzymes for this purpose include, forexample, E. coli DNA polymerase I, Klenow fragment of E coli DNApolymerase I, T4 DNA polymerase, other available DNA polymerases,polymerase muteins, reverse transcriptase, and other enzymes, includingheat-stable enzymes (i.e., those enzymes which perform primer extensionafter being subjected to temperatures sufficiently elevated to causedenaturation). Suitable enzymes will facilitate combination of thenucleotides in the proper manner to form the primer extension productswhich are complementary to each receptor PTK nucleic acid strand.Generally, the synthesis will be initiated at the 3′ end of each primerand proceed in the 5′ direction along the template strand, untilsynthesis terminates, producing molecules of different lengths. Theremay be agents for polymerization, however, which initiate synthesis atthe 5′ end and proceed in the other direction, using the same process asdescribed above.

[0053] The newly synthesized receptor PTK strand and its complementarynucleic acid strand will form a double-stranded molecule underhybridizing conditions described above and this hybrid is used insubsequent steps of the process. In the next step, the newly synthesizeddouble-stranded molecule is subjected to denaturing conditions using anyof the procedures described above to provide single-stranded molecules.

[0054] The above process is repeated on the single-stranded molecules.Additional agent for polymerization, nucleotides, and primers may beadded, if necessary, for the reaction to proceed under the conditionsprescribed above. Again, the synthesis will be initiated at one end ofeach of the oligonucleotide primers and will proceed along the singlestrands of the template to produce additional nucleic acid. After thisstep, half of the extension product will consist of the specific nucleicacid sequence bounded by the two primers.

[0055] The steps of denaturing and extension product synthesis can berepeated as often as needed to amplify the receptor PTK nucleic acidsequence to the extent necessary for detection. The amount of thespecific nucleic acid sequence produced will accumulate in anexponential fashion.

[0056] Sequences amplified by the methods of the invention can befurther evaluated, detected, cloned, sequenced, and the like, either insolution or after binding to a solid support, by any method usuallyapplied to the detection of a specific DNA sequence such as PCR,oligomer restriction (Saiki, et al., Bio/Technology, 3:1008-1012, 1985),allele-specific oligonucleotide (ASO) probe analysis (Conner, et al.,Proc. Natl. Acad. Sci. USA, 80:278, 1983), oligonucleotide ligationassays (OLAs) (Landegren, et al., Science, 241:1077, 1988), and thelike. Molecular techniques for DNA analysis have been reviewed(Landegren, et al., Science, 242:229-237, 1988).

[0057] The present invention also provides methods for the treatment ofdisease employing gene therapy that modulates cellular differentiationor maturation. Such therapy can be affected by introduction ofpolynucleotide sequences of the invention into cells of a subject havinga disease. Delivery of polynucleotide can be achieved using techniqueswell known in the art. For example, a recombinant expression vector,such as a chimeric virus, or a colloidal dispersion system can beemployed.

[0058] Various viral vectors which can be utilized for introduction ofpolynucleotide according to the present invention, include adenovirus,herpes virus, vaccinia, or, preferably, an RNA virus such as aretrovirus. Preferably, the retroviral vector is a derivative of amurine or avian retrovirus. Examples of retroviral vectors in which asingle foreign gene can be inserted include, but are not limited to:Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can incorporate a gene for aselectable marker so that transduced cells can be identified andgenerated.

[0059] By inserting a polynucleotide encoding the receptor PTK ofinterest into a viral vector, along with another gene which encodesligand for a receptor on a specific target cell, the vector now becomestarget specific. Retroviral vectors can be made target specific byincluding in the retroviral vector a polynucleotide encoding a targetrelated binding substance. Preferred targeting is accomplished by usingan antibody to target the retroviral vector. Those of skill in the artwill know of, or can readily ascertain without undue experimentation,specific polynucleotide sequences which can be inserted into theretroviral genome to allow target specific delivery of the retroviralvector containing the receptor PTK polynucleotide.

[0060] Since recombinant retroviruses are defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided, for example, by using helper cell lines thatcontain plasmids encoding all of the structural genes of the retrovirusunder the control of regulatory sequences within the LTR. These plasmidsare missing a nucleotide sequence which enables the packaging mechanismto recognize an RNA transcript for encapsidation. Helper cell lineswhich have deletions of the packaging signal include, but are notlimited to, Ψ2, PA317 and PA12, for example. These cell lines produceempty virions, since no genome is packaged. If a retroviral vector isintroduced into such cells in which the packaging signal is intact, butthe structural genes are replaced by other genes of interest, the vectorcan be packaged and vector virion produced. The vector virions producedby this method can then be used to infect a tissue cell line, such asNIH 3T3 cells, to produce large quantities of chimeric retroviralvirions.

[0061] Alternatively, NIH 3T3 or other tissue culture cells can bedirectly transfected with plasmids encoding the retroviral structuralgenes gag, pol and env, by conventional calcium phosphate transfection.These cells are then transfected with the vector plasmid containing thegenes of interest. The resulting cells release the retroviral vectorinto the culture medium.

[0062] Another targeted delivery system for introduction ofpolynucleotides encoding the receptor PTKs of the invention is acolloidal dispersion system. Colloidal dispersion systems includemacromolecular complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome.

[0063] Since the receptor PTK polypeptide may be indiscriminate in itsaction with respect to cell type, a targeted delivery system offers asignificant improvement over randomly injected non-specific liposomes. Anumber of procedures can be used to covalently attach either polyclonalor monoclonal antibodies to a liposome bilayer. Antibody-targetedliposomes can include monoclonal or polyclonal antibodies or fragmentsthereof such as Fab, or F(ab′)₂, as long as they bind efficiently to anepitope on the target cells. Liposomes may also be targeted to cellsexpressing receptors for hormones or other serum factors.

[0064] Liposomes are artificial membrane vesicles which are useful as invitro and in vivo delivery vehicles. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2-4.0 μm canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA, DNA, intact virions and peptides can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends Biochem. Sci., 6:77,1981). In order for a liposome to be an efficient transfer vehicle, thefollowing characteristics should be present: (1) encapsulation ofpolynucleotides of interest at high efficiency without compromisingbiological activity; (2) preferential and substantial binding to targetcells relative to non-target cells; (3) delivery of aqueous contents ofvesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information (Mannino, etal., Biotechniques, 6:682, 1988).

[0065] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting receptor in stable association withthe liposomal bilayer. Various linking groups can be used for joiningthe lipid chains to the targeting receptor.

[0066] In general, the targeted delivery system will be directed to cellsurface receptors thereby allowing the delivery system to find and “homein” on the desired cells. Alternatively, the delivery system can bedirected to any cell surface molecule preferentially found in the cellpopulation for which treatment is desired and capable of associationwith the delivery system. Antibodies can be used to target liposomes tospecific cell-surface molecules. For example, where a tumor isassociated with a receptor PTK of the invention, certain antigensexpressed specifically or predominantly on the cells of the tumor may beexploited for the purpose of targeting antibody tyrosine kinase receptorDNA-containing liposomes directly to a malignant tumor, if desired.

[0067] An alternative use for recombinant retroviral vectors comprisesthe introduction of polynucleotide sequences into the host by means ofskin transplants of cells containing the virus. Long term expression offoreign genes in implants, using cells of fibroblast origin, may beachieved if a strong housekeeping gene promoter is used to drivetranscription. For example, the dihydrofolate reductase (DHFR) genepromoter may be used. Cells such as fibroblasts, can be infected withvirions containing a retroviral construct containing the receptor PTKgene of interest together with a gene which allows for specifictargeting, such as a tumor-associated antigen and a strong promoter. Theinfected cells can be embedded in a collagen matrix which can be graftedinto the connective tissue of the dermis in the recipient subject. Asthe retrovirus proliferates and escapes the matrix it will specificallyinfect the target cell population. In this way the transplantationresults in increased amounts of receptor PTK being produced in cellsmanifesting the disease.

[0068] Because the present invention identifies nucleotide sequencesencoding novel receptor PTKs, it is possible to design therapeutic ordiagnostic protocols utilizing these sequences. Thus, where a disease isassociated with a receptor PTK of the invention, the polynucleotidesequence encoding the PTK can be utilized to design sequences whichinterfere with the function of the receptor. This approach utilizes, forexample, antisense nucleic acid and ribozymes to block translation ofspecific receptor mRNA, either by masking the mRNA with antisensenucleic acid or by cleaving it with ribozyme.

[0069] Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule(Weintraub, Scientific American, 262:40, 1990). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA since the cell will not translate a mRNA that isdouble-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely tocause problems than larger molecules when introduced into the targetreceptor-producing cell. The use of antisense methods to inhibit the invitro translation of genes is well known in the art (Marcus-Sakura,Anal.Biochem., 172:289, 1988).

[0070] Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

[0071] There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

[0072] Antisense sequences can be therapeutically administered bytechniques as described above for the administration of receptor PTKpolynucleotides. Targeted liposomes are especially preferred fortherapeutic delivery of antisense sequences.

[0073] The following Examples are intended to illustrate, but not tolimit the invention. While such Examples are typical of those that mightbe used, other procedures known to those skilled in the art mayalternatively be utilized.

EXAMPLE 1 Isolation of Novel PTK Clones

[0074] PCR was used to amplify PTK-related sequences located between thedegenerate oligonucleotide primer sequences shown in TABLE 1. Theseprimers correspond to the amino acid sequences HRDLAAR (SEQ ID NO:27)(upstream) and DVWS(F/Y)G(I/V) (SEQ ID NO:28) (downstream), which flanka highly conserved region of the kinase domain shared by receptor PTKs(Hanks, et al., Science, 241:42-52, 1988). The upstream primer waschosen to exclude members of the src family of cytoplasmic tyrosinekinases. The downstream primer was chosen such that a second highlyconserved amino acid sequence diagnostic or PTKs—P(I/V)(K/R)W(T/M)APE(SEQ ID NO:29)—would be contained within amplified PCR products.

[0075] The DNA substrates used for amplification were sciatic nerve cDNApopulations prepared for use in the construction of subtracted cDNAlibraries. Three different subtracted cDNAs were produced. The firsttwo, UN and TWI, were enriched for transcripts expressed predominantlyin Schwann cells. The third, BD, was enriched for transcripts sharedbetween Schwann cells and myelinating stage (P17-23) brain. Two initialhybridizations were performed. Both samples contained 500 ng ofsingle-stranded sciatic nerve cDNA mixed with the followingpoly(A)-selected RNAs: 10 μg of muscle, 7.5 μg of liver, and 5 μg ofkidney. Both samples also contained a series of RNAs synthesized invitro; these encoded portions of the sense strand of the followingSchwann cell transcripts: NGF receptor, glial fibrillary acidic protein,proteolipid protein, protein zero, myelin basic protein, and CNPase. Thefirst sample contained, in addition, 10 μg of poly(A)-selected RNA fromrat brain cerebellum (P19) and cortex (P3). Each hybridization wasallowed to proceed to approximately R₀t 2000. Following hybridization,these samples were bound to hydroxylapatite (0.12M phosphate buffer, 65°C.). For the first sample, material not binding to hydroxylapatite (HAP)was collected and converted to a double-stranded form. This material wasdesignated UN (unbound). For the second sample, cDNA not binding to thecolumn was further hybridized with 40 μg of poly(A)-selected RNA fromrat cerebellum (equal mix of P17 and P23) until R₀t 800. This mixturewas re-applied to hydroxylapatite. The unbound material was collectedand converted to a double-stranded form and designated TWI (twiceunbound). The material that bound to the HAP column was then eluted andalso converted to a double-stranded DNA form. This fraction was calledBD (bound).

[0076] Approximately 2-4 ng of the UN and TWI subtracted cDNAs and 1 ngof the BD cDNA were used in each of the amplifications, which wereconducted using reagents and instructions provided by U.S. Biochemicals.The final concentration of magnesium ion was increased to 2.1 mM.Thirty-nine cycles of amplification were performed on a water-cooledvtwb Model 1 cycler (San Diego, Calif.). Amplification parametersincluded an initial 1 minute denaturation step at 94° C., a 5 minuteannealing at 37° C., a 5 minute extension at 65° C., and a 0.3 minutedenaturation at 94° C. Approximately 4 μg of each of the degenerateprimers (TABLE 1) was included in each amplification. The unusually lowannealing temperatures employed in these amplifications may favorpolymerase extension from stably-hybridized oligonucleotide primers,resulting in a broader and less-biased amplified population than thoseobtained with previous protocols (Wilks, Proc. Natl. Acad. Sci. USA,86:1603-1607, 1989). TABLE 1 (SEQ ID NOS:30-35)

[0077] Amplified DNAs were size fractionated on 5% non-denaturingacrylamide gels. The gels were stained with ethidium bromide (1 μg/ml)and amplified bands of ˜220 bp were excised. These bands were elutedovernight into 0.5 M ammonium acetate. 1 mM EDTA, 0.2% SDS, and elutedDNA was then precipitated with 10 μg of tRNA carrier. Recovered PCRproducts were blunt-ended using T4 DNA polymerase, and phosphorylatedusing T4 polynucleotide kinase. Approximately 40 ng of insert was thenligated with 200 ng of dephosphorylated SmaI/EcoRV-digested pBluescriptplasmid. One-tenth of each ligation was used to transform MC1061bacteria.

[0078] The DNA sequence of both strands of each PCR product subclone wasdetermined from alkaline lysis miniprep DNA, using the dideoxy chaintermination method. In those cases in which clones having apparentlyidentical inserts were isolated multiple times, the sequence ofcomplementary strands was derived from independent clones.

EXAMPLE 2 Sequence Analysis of PCR Subclones

[0079] Sequence analysis of 168 PCR product subclones yielded 155 withsignificant similarity to the tyrosine kinase family. TABLE 2 lists the27 distinct kinase domain sequences contained in this set, whichincludes those of the abl (human; Shtivelman, et al., Cell, 47:277-284,1986) arg (human: Kruh, et al., Science, 234:1545-1548, 1986), and fercytoplasmic (nonreceptor) kinases (human, Hao, et al., Mol. Cell. Biol.,9:1587-1593, 1989), as well as those of the receptors for EGF-R (human;Ullrich, et al., Nature 309, 418-425, 1984), PDGF-A (human; Matsui, etal., Science, 243:800-804, 1989; rat; Lee, et al., Science, 245:57-60,1989; Reid, et al., Proc. Natl. Acad. Sci. USA, 87:1596-1600, 1990;Safran, et al., Oncogene, 5:635-643, 1990), colony-stimulating factor 1(CSF-1; human; Coussens, et al., Nature, 320:277-280, 1986; mouse;Rothwell and Rohrschneider, Oncogene Res., 1:311-324, 1987, andinsulin-like growth factor 1 (IGF-1; human; Ullrich, et al., EMBO, J.,5:2503-2512, 1986).

[0080] Other domain sequences listed include fes (human; Roebroek, etal., EMBO J., 4:2897-2903, 1985); Dsrc (Drosophila; Gregory, et al.,Mol. Cell. Biol., 7:2119-2127, 1987); eph (human; Hirai, et al.,Science, 238:1717-1720, 1987; eck (human; Lindberg and Hunter, Mol.Cell. Biol., in press, 1990); elk (rat; Letwin, et al., Oncogene,3:621-627, 1988); neu (Bargmann, et al., Nature, 319:226-230, 1986); bek(mouse; Kornbluth, et al., Mol. Cell. Biol., 8:5541-5544, 1988); flt(human; Shibuya, et al., Oncogene, 5:519-524,1990); trk, (human;Martin-Zanca, et al., Nature, 319:743-748, 1986), and trk B (mouse;Klein, et al., EMBO J., 8:3701-3709, 1989).

[0081] Amino acid sequences were deduced from the nucleotide sequence ofthe 27 different PTK domain cDNAs. Deduced amino acid sequencescorresponding to the oligonucleotide primers used for PCR amplificationwere not included. Kinase domain sequences are segmented according tothe subdomains defined by Hanks, et al. (Science, 241:42-52, 1988).After each sequence is a number indicating the number of times it wasidentified. Numbers listed parenthetically correspond to clones uniquelyobtained from amplification of the BD substrate. The segregation ofkinase domain subfamilies is based solely on amino acid sequenceconservation; sequences denoted by an asterisk were not encountered inthis survey but have been included to facilitate comparisons.

[0082] The high percentage of isolates encoding tyrosine kinases (92%)and the large number of different kinase clones obtained probablyreflect the highly degenerate primers and low temperature annealing andextension parameters used for PCR amplification, as well as thestringent size criteria used in the subcloning and sequencing of PCRproducts.

[0083] Of the 27 different kinases identified in this nervous systemsurvey, 11 (tyro-1 through tyro-8 and tyro-10 through tyro-12) arenovel. For the previously identified kinases, several rat isolatesdiffer by 1 or 2 amino acids from the kinase domain sequences reportedfor other species. Nucleotide sequence comparisons suggest that thesedifferences are accounted for by species variation and do not representthe amplification of novel kinase cDNAs. The novel isolates tyro-1 andtyro-11 were each obtained only a single time.

[0084] The kinase domain sequences of tyro-1 through tyro-13 have beengrouped by similarity to the equivalent sequences of other PTKs (TABLE2). The indicated subfamilies were defined with reference to acomputer-generated phylogenetic tree, constructed from an analysis of 13novel partial PTK sequences along with a set of 55 additional PTKs,according to the methods of Fitch and Margoliash (Science, 15:279-284,1967) as implemented by the programs of Feng and Doolittle (J. Mol.Evol., 25:351-360, 1987). The resulting closely related sequenceclusters were used to organize the kinase subfamilies presented in TABLE2. Tyro-1 and tyro-4, for example, are related to the epithelial cellkinase (eck) (Lindberg and Hunter, Mol. Cell. Biol., in press, 1990),tyro-2 to the EGF receptor and the neu proto-oncogene (Bargmann, et et.,Nature, 319:226-230, 1986), tyro-5, tyro-6, and tyro-11 to the elkkinase (Letwin, et al., Oncogene, 3:621-627, 1988), tyro-9 to the bFGFreceptor, and tyro-10 to trk and trkB (Martin-Zanca, et al., Nature,319:743-748, 1986; Klein, et al., EMBO J., 8:3701-3709, 1989). Althoughthey exhibit similarity to the insulin receptor, tyro-3, tyro-7, andtyro-12 are listed as a novel subfamily since they are more closelyrelated to each other than to any previously described kinase. The eck-and elk-related sequences are listed in separate subsets, but it isimportant to note the high degree of similarity between thesesubfamilies. The sequences of fes, trk, trkB, and Dsrc28 (each markedwith an asterisk) are included in TABLE 2 only for comparison, sincethey were not encountered in these cloning studios.

EXAMPLE 3 Tissue Expression Profile of Novel PTK mRNAs

[0085] The expression pattern of the 13 novel kinase clones werecharacterized by first examining the relative levels of mRNA present ina variety of neonatal and adult rat tissues. Radiolabeled cDNA probesfor each of these clones, as well as probes prepared from isolates ofthe bFGF receptor, bek, and elk kinases, were hybridized to a set ofeight parallel Northern blots containing RNA isolated from kidney,liver, spleen, heart, skeletal muscle, brain, sciatic nerve, andcultured Schwann cells. RNA was isolated from Schwann cells cultured inboth the presence and absence of the adenylate cyclase activatorforskolin, since at least one receptor PTK gene (that encoding thePDGF-B receptor) exhibits ell-specific cAMP induction in these cells(Weinmaster and Lemke, EMBO J., 9:915-920, 1990) individual blots werein some cases reutilized for as many as four rounds of hybridization.

[0086] Total RNA from various tissues was prepared by the method ofChomczynski and Sacchi (Anal. Biochem., 162:156-159, 1987). Oneadditional phenol-chloroform extraction was performed prior to nucleicacid precipitation. Poly(A)-selected RNA samples were purified by eithercolumn chromatography or in batch using oligo(dT)-cellulose type III(Collaborative Research). RNA samples were denatured in 50% formamide,2.2M formaldehyde, and MOPS at 65° C. for 10 min, electrophoresed in1.0% agarose, 2.2M formaldehyde, and MOPS, transferred to Nytran filters(Schleicher & Schuell) and baked at 80° C. for 2 hr as previouslydescribed (Monuki, et al., Neuron, 3:783-793, 1989). Probes for blothybridizations were prepared using [α-³²P] dCTP and a random hexamerpriming kit, according to instructions provided by the manufacturer(Bethesda Research Laboratories). In all cases, final wash stringencyfor Northern blots was set at 0.2×SSC, 0.2% SDS, 65 ° C.

[0087] In situ hybridization was performed according to Simmons, et al.(J. Histotechnology, 12:169-181, 1989), with minor modifications.Paraformaldehyde-fixed brain sections (30 μm), from either adult or33-day-old rats were used. Antisense probes from PCR product Subcloneswere prepared using 125 μCi or [³⁵S] UTP (1250 Ci/mmol: New EnglandNuclear) in a 10 μl transcription reaction, with reagents obtained fromStratagene (La Jolla, Calif.). Hybridizations were performed at 55° C.for 22 hr using approximately 75 μl or 5×10⁶ cpm/ml probe per slide.RNAase A digestions were performed in buffer prewarmed to 37° C. Thefinal wash stringency was 0.1×SSC at 60° C. for 35 min. Emulsion-dippedslides were exposed for 2 weeks prior to developing. Slides werecounterstained with thionin.

[0088] The various tissue expression profiles are shown in FIG. 1.Poly(A) (left 10 lanes) or total RNA (tot, right 4 lanes) from theindicated rat tissues was analyzed for expression of PTK mRNAs. Alltissues were taken from animals 27 days postnatal, except whereotherwise indicated. Sciatic nerves (sciatic) were obtained from7-to-8-day-old rats. Rat Schwann cells were cultured in either thepresence (+) or absence (−) of 20 μM forskolin for 48 hr prior toharvesting. All lanes contain either 2.5 μg of poly(A)⁺ RNA or 10 μg oftotal RNA, except for the cultured Schwann cell poly(A)⁺ lanes, whichcontain 1.0 μg each. The relative migration of 18S and 28S ribosomalRNAs; as determined by methylene blue staining, is indicated by thearrowheads. Filters 1-13 show hybridization with ³²P radiolabeled cDNAprobes to tyro-1 through tyro-13. Also shown for comparison is thehybridization observed using isolates of elk, the bFGF receptor (FGFR),and the bek FGF receptor. Exposure times were as follows: 34 hr (1, 5,6, 7, 11), 41 hr (3, 4, FGFR), 120 hr (2, 9, 10, bek), 158 hr (8, 13,elk), 8 days (12).

[0089] The results of this analysis (FIG. 1) demonstrate that 6 of the11 novel kinase genes (tyro-1 through tyro-6), together with the elkgene, are preferentially expressed by cells of the nervous system. Forexample, tyro-1, a novel member of the eck kinase subfamily, exhibitedstrong hybridization to brain mRNA, a faint signal in Schwann cells, andvery faint signals in kidney and heart. Tyro-4 also a novel member ofthe eck subfamily, exhibited more modest hybridization to two mRNAs inpostnatal day 5(P5) brain, with lower signals evident in older brains aswell as kidney and heart. The novel EGF receptor homolog tyro-2identified a high molecular weight mRNA in brain that could also bedetected in kidney and heart. It is possible that the very low tyro-1,tyro-2, and tyro-4 hybridization signals observed in kidney and heartare due to neural contamination from the adrenal gland and cardiacganglia, respectively. Tyro-3, a member of the novel kinase subfamilywith similarity to the insulin receptor, showed intense hybridization tobrain mRNA, with very faint signals in perhaps all other tissues.

[0090] Members of the same receptor-configured kinase subfamilyoccasionally exhibited very different patterns of expression. Within theelk subfamily, for example, elk itself and the related kinases tyro-5and tyro-6 were exclusively or predominantly expressed in neuraltissues, elk strongly hybridized to two mRNA species in brain andSchwann cells, tyro-5 exhibited strong hybridization to P5 brain mRNAwith reduced signals present in later stage brains and in Schwann cells,and tyro-6 gave a strong hybridization signal in cultured Schwann cells,weaker signals in brain, and very faint but detectable signals in othertissues. In contrast, expression of the elk-related kinase tyro-11 waspredominant in heart and kidney, but expressed at lower levels in neuraltissue. The distinct hybridization patterns observed between members ofthis closely related subfamily indicate that despite significantsimilarity at the nucleotide level, cross-hybridization is not readilydetected when hybridizations are carried out at high stringency. Tyro-5and tyro-6, the most closely related of the PTK domains we analyzed,exhibit 84.2% nucleotide identity over the kinase domain, but theirhybridization profiles can be readily distinguished (FIG. 1, compareprofiles 5 and 6).

[0091] Among those kinases not restricted to neural cells, tyro-9, amember of the FGF receptor subfamily, exhibited a pattern of expressionthat was distinct from that of either the bFGF receptor or bek. Moststrongly expressed in kidney and liver, it exhibited only weakhybridization signals with brain mRNA. At two extremes of expression,tyro-12 yielded weak hybridization signals in all tissues, withexpression being somewhat lower in heart and muscle, but tyro-8(distantly related to Dsrc28) yielded only an extremely faint signal inspleen and heart.

[0092] Schwann cell expression of certain kinase genes was stronglyregulated by cAMP (FIG. 1). As for the PDGF receptor gene (Weinmasterand Lemke, EMBO J., 9:915-920, 1990), expression of the elk and FGFreceptor genes was significantly up-regulated by 48 hr treatment withforskolin. Since cAMP induction of the PDGF receptor appears to accountfor the synergistic effect on Schwann cell proliferation achieved withcombined application of PDGF and forskolin (Weinmaster and Lemke, EMBOJ., 9:915-920, 1990), cAMP induction of the FGF receptor may alsoexplain the similar synergistic effect observed for the combination ofFGF and forskolin (Davis and Stroobant, J. Cell Biol., 110:1353-1360,1990). Importantly, cAMP induction was not observed for most of thereceptor PTKs expressed by Schwann cells; the tyro-1, tyro-3, tyro-6,tyro-7, tyro-12, and tyro-13 mRNAs were down-regulated in the presenceof forskolin, and expression of the tyro-5 and tyro-11 genes was notaffected by the drug.

[0093] Several receptor PTKs exhibited relatively modest signals insciatic nerve compared with cultured Schwann cells or other tissues.This is probably a function of both the cellular heterogeneity of thenerve, which contains a substantial number of fibroblasts andendothelial cells, and the great sensitivity of PCR amplification.

EXAMPLE 4 Developmental Expression Profile of Neural PTK mRNAs

[0094] Since many of the determinative events in mammalian neuraldevelopment occur near the midpoint of embryogenesis, a study wasperformed to determine whether any of the novel neural kinase genes wereexpressed embryonically. To assess their developmental expression, a setof Northern blots containing mRNA isolated from the brains of ratsranging in age from embryonic day 12 (E12) to adult were probed. Forcomparison, included were the bFGF receptor and elk in this survey, theresults of which are presented in FIG. 2. For each of the novel kinasegenes, expression was observed in the developing central nervous systemat E12, a time at which multiple influences on both neural cellproliferation and differentiation are known to be exercised.

[0095] Poly(A)⁺ RNA (2 μg) from rat brains obtained from animals of theindicated ages (E12 to 7 months postnatal) was analyzed for theexpression of PTK mRNAs. Filters 1-6 show hybridization obtained with³²P-radiolabled cDNA probes to tyro-1 through tyro-6. Also shown are thehybridization profiles obtained using isolates of elk and the bFGFreceptor (FGFR). The relative migration of 18S and 28S ribosomal RNAs,as determined by methylene blue staining, is indicated by thearrowheads. Exposure times are as follows: 15 hr (1, 3, 5, elk, FGFR),22 hr (4, 6), 50 hr (2).

[0096] Although detected in adult brain, three of the novel kinase geneswere maximally expressed embryonically. mRNA encoding the elk-relatedkinase tyro-6, for example, was most abundantly expressed at E12;expression gradually fell until P10 and was relatively constantthereafter. Similarly, mRNA encoding the closely related kinase tyro-5was maximally expressed at E14; expression fell sharply after P5 to amuch lower steady-state level in the adult brain. The gene encoding theeck-related kinase tyro-4 exhibited a similar, though even more dramaticregulation, with a peak in expression at E14/17, a sharp drop at birth,and a low steady-state level after P10.

[0097] In contrast to the pronounced drop in expression for tyro-4 andtyro-5, expression of mRNA encoding the eck-like kinase tyro-1, whileexhibiting some temporal fluctuation, was relatively constant throughoutneural development. A similar, though less variable-developmentalprofile, was observed for mRNA encoding the bFGF receptor. Althoughmaximal expression was observed at E12, bFGF receptor mRNA levels fellonly modestly during the course of brain development and remained highin adult animals. Of the novel kinase genes analyzed in FIG. 2, onlytyro-3 exhibited a significant increase in expression during late neuraldevelopment, with appreciably higher mRNA levels (relative to E12)evident after P20.

EXAMPLE 5 In situ Localization of Novel PTK Transcripts in Brain

[0098] To determine whether any of the novel neural kinases exhibitedcell type-restricted expression in the vertebrate central nervoussystem, radiolabeled antisense RNA probes for each of the clones wereprepared and these probes hybridized in situ to 30 μm brain sectionsprepared from 33-day-old and adult male rats. For comparison, antisenseprobes prepared from our isolates of the bFGF receptor and the relatedFGF receptor bek were included.

[0099] Although the profiles of these brain sections represented aselective sampling of the brain, they nonetheless demonstrated thatexpression of each of the novel neural kinases is highly regionalized.Tyro-1 mRNA was the most widely expressed in adult brain. Tyro-1 probesexhibited exceptionally strong and continuous hybridization in allfields of the hippocampus and the dentate gyrus and throughout theneocortex, with a diffuse band present in layer 3. Strong hybridizationwas also seen in the Purkinje cell layer, the inferior olive, andlateral nucleus of the cerebellum, but not in the cerebellar granulecell layers.

[0100] In contrast, the tryo-2 gene exhibited a much more restrictedpattern of expression. Hybridization was again evident throughout allfields of the hippocampus and the dentate gyrus, but signals wererestricted to occasional (˜1 in 10) cells. This striking, punctatepattern of hippocampal hybridization was not seen for any other PTKgene. A similarly restricted pattern of tyro-2 hybridization was alsoobserved throughout the neocortex. Stronger and more continuoushybridization was evident in the medial habenula and in the reticularnucleus of the thalamus, but in contrast to tyro-1, no signal abovebackground was observed in the remainder of the thalamus. The strongesttyro-2 hybridization signal in the brain was observed in an intercalatednucleus of the amygdala. No signal was evident in the Purkinje celllayer in the cerebellum. The hybridization pattern have observed fortyro-2 is largely consistent with its expression by a subset of7-amino-n-butyric acid (GABA)-ergic neurons.

[0101] In situ hybridization signals corresponding to tyro-3 mRNApresented an equally striking pattern. In the hippocampus, stronghybridization was observed in the CA1 field. However, upon crossing theborder from CA1 to the shorter CA2 field an abrupt drop in the tyro-3hybridization signal was observed. The tyro-3 signal remained muchreduced in CA3 (relative to CA1), and no signal at all in the dentategyrus was observed. Tyro-3 therefore provides an excellent molecularmarker for the CA1/CA2 transition, previously defined on the basis ofhippocampal cell size and circuitry. Robust tyro-3 hybridization wasalso evident in large cells throughout neocortex, with the strongestsignals being observed in deeper layers. In the cerebellum, stronghybridization was observed to granule cells, but not to Purkinje cells,a pattern that was the opposite of that observed for tyro-1.

[0102] Consistent with their developmental expression profiles, tyro-4,tyro-5, and tyro-6 exhibited the most restricted patterns of expressionin adult brain. Distinct hybridization to tyro-4 was evident in thefacial nucleus of the pons, with more modest signals present in the bednucleus of the anterior commissure and the triangular nucleus of theseptum. The tyro-5 gene was expressed weakly in cortex, at a modestlevel in all fields of the hippocampus, and in a subset of Purkinjecells in the cerebellum. The tyro-6 gene showed a similar pattern ofexpression, giving a signal in Purkinje cells and weak signals in thehippocampus.

[0103] The two FGF receptor genes examined, those encoding the bFGFreceptor and bek, exhibited very different patterns of expression in thebrain. mRNA encoding the bFGF receptor was expressed at high levels inhippocampal neurons, but exhibited a field distribution that was nearlythe inverse of tyro-3., i.e., expression was reduced in CA1 relative toCA2 and CA3. mRNA levels in the dentate gyrus were lowest of all. Theexpression of bFGF receptor mRNA in the choroid plexus and in thecentral nucleus of the amygdala and in a narrow band of cells in layer 6of neocortex, a region not seen in the previous work of Wanaka, et al.(Neuron, 5:267-281, 1990) was also observed. In contrast, expression ofbek mRNA was largely confined to non-neuronal cells. High levelexpression was observed in the choroid plexus, and in the white matterglia of the cerebellum and the pons. Diffusely localized hybridizationto a layer of cells that may be Bergmann glia was also apparent in thecerebellum. The cerebellar expression pattern of bek was clearlydistinct from the patterns observed for tyro-5 and tyro-6, which markedPurkinje cells, but exhibited no hybridization to white matter glia.TABLE 2 (SEQ ID NOS.:36-54) KINASE DEDUCED AMINO ACID SEQUENCES FORPUTATIVE TYROSINE KINASES SUB-FAMILY VI VII VIII IX INCIDENCE abl ablNCLVGENH LVKVADFGLSRLMTGDTYTAH AGAKFPIKWTAPESL AYNFKSIKS  6 arg NCLVGENHVVKVADFGLSRLMTGDTYTAH AGAKFPIKWTAPESL AYNFKSIKS  3 fes/fps* NCLVTEKNVLKISDFGMSREEADGVYAASG GLRQVPVKWTAPEAL NYGRYSSES fer NCLVGENNTLKISDFGMSRQEDGGVYSSS GLKQIPIKWTAPEAL NYGRYSSES  2 src Dsrc28* NCLVGSENVVKVADFGLARYVLDDQYTSSG GTKFPIKWAPPEVL NYTRFSSKS tyro-8 NCLVDSDLSVKVSDFGMTRYVLDDQYVSSV GTKFPVKWSAPEVF HYFKTSSKS  2 tyro-13 tyro-13NVLVSEDN VAKVSDFGLTKEASSTQ DTGKLPVKWTAPEAL REKKFSTKS 11 eph/eck/elk eph*NILVNQNL CCKVSDFGLTRLLDDFDGTYET QGGKIPIRWTAPEAI AHRIFTTAS eck NILVNSNLVCKVSDFGLSRVLEDDPEATYTT SGGKIPIRWTAPEAI SYRKFTSAS  5 tyro-1 NILVNSNLVCKVSDFGMSRVLEDDPEAAYTT RGGKIPIRWTAPEAI AYRKFTSAS  1 tyro-4 NILINSNLVCKVSDFGLSRVLEDDPEAAYTT RGGKIPIRWTSPEAI AYRKFTSAS  4 elk NILVNSNLVCKVSDFGLSRYLQDDTSDPTYTSS LGGKIPVRWTAPEAI AYRKFTSAS  1 tyro-5 NILVNSNLVCKVSDFGLSRFLEDDTSDPTYTSA LGGKIPIRWTAPEAI QYRKFTSAS (3) tyro-6 NILVNSNLVCKVSDFGLSRFLEDDPSDPTYTSS LGGKIPIRWTAPEAI AYRKFTSAS  3 tyro-11 NILVNSNLVCKVSDFGLSRFLEENSSDPTYTSS LGGKIPIRWTAPEAI AFRKFTSAS (1) EGF-R EGF-RNVLVKTPQ HVKITDFGLAKLLGAEEKEYHA EGGKVPIKWMALESI LHRIYTHQS  3 neuNVLVKSPN HVKITDFGLARLLDIDETEYHA DGGKVPIKWMALESI LRRRFTHQS 10 tyro-2NVLVKSPN HVKITDFGLARLLEGDEKEYNA DGGKMPIKWMALECI HYRKFTHQS  8 FGF-RbFGF-R NVLVTEDN VMKIADFGLARDIHHLDYYKKT TNGRLPVKWMPEAL FDRIYTHQS  4 bekNVLVTENN VMKIADFGLARDINNIDYYKKT TNGRLPVKWMAPEAL FDRVYTHQS  2 tyro-9NVLVTEDD VMKIADFGLARGVHHIDYYKKT SNGRLPVKWMAPEAL FDRVYTHQS  1 PDGF-RPDGF-AR NVLLAQGK IVKICDFGLARDIMHDSNYVSK GSTFLPVKWMAPESI FDNLYTTLS  3PDGF-BR NMLICEGK LVKICDFGLARDIMRDSNYISK GSTFLPLKWMAPESI FNSLYTTLS  1CSF-1R NVLLTSGH VAKIGDFGLARDIMNDSNYVVK GNARLPVKWMAPESI FDCVYTVQS 20 fltNLLLSENN VVKICDFGLARDIYKNPDYVRR GDTRLPLKWMAPESI FDKVYSTKS  5 tyro-3tyro-3 NCMLAEDM TVCVADFGLSRKIYSGDYYRQG CASKLPVKWLALESL ADNLYTVHS  3tyro-7 NCMLNENM SVCVADFGLSKKIYNGDYYRQG PFAKMPVKWIAIESL ADRVYTSKS  4tyro-12 NCMLRDDM TVCVADFGLSKKIYSGDYYRQG RIAKMPVKWIAIESL ADRVTYSKS (3)Insulin-R trk* NCLVGQGL VVKIGDFGMSRDIYSTDYYRVG GRTMLPIRWMPPESI LYRKETTEStrkB* NCLVGENL LVKIGDFGMSRDVYSTDYYRVG GHTMLPIRWMPPESI MYRKFTTES IGF1RNCMVAEDF TVKIGDFGMTRDIYETDYYRKG GKGLLPVRWMSPESL KDGVFTTHS  2 tyro-10NCLVGKNY TIKIADFGMSRNLYSGDYYRIQ GRAVLPIRWMSWESI LLGKFTTAS (6)

[0104] The foregoing is meant to illustrate, but not to limit, the scopeof the invention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

1 54 165 base pairs nucleic acid single linear DNA Tyro-1 CDS 1..165 1AAC ATT CTG GTA AAC AGC AAC TTG GTC TGC AAG GTG TCT GAT TTC GGC 48 AsnIle Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15ATG TCC AGG GTG CTT GAG GAT GAC CCG GAA GCA GCC TAT ACT ACC AGG 96 MetSer Arg Val Leu Glu Asp Asp Pro Glu Ala Ala Tyr Thr Thr Arg 20 25 30 GGCGGC AAG ATT CCC ATC CGG TGG ACT GCA CCA GAA GCA ATT GCG TAT 144 Gly GlyLys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ala Tyr 35 40 45 CGT AAATTT ACC TCA GCC AGT 165 Arg Lys Phe Thr Ser Ala Ser 50 55 55 amino acidsamino acid linear protein 2 Asn Ile Leu Val Asn Ser Asn Leu Val Cys LysVal Ser Asp Phe Gly 1 5 10 15 Met Ser Arg Val Leu Glu Asp Asp Pro GluAla Ala Tyr Thr Thr Arg 20 25 30 Gly Gly Lys Ile Pro Ile Arg Trp Thr AlaPro Glu Ala Ile Ala Tyr 35 40 45 Arg Lys Phe Thr Ser Ala Ser 50 55 2437base pairs nucleic acid single linear DNA Tyro-2 CDS 3..2118 3 CA AACTGT GTG GAG AAA TGT CCA GAT GGC CTA CAG GGA GCA AAC AGT 47 Asn Cys ValGlu Lys Cys Pro Asp Gly Leu Gln Gly Ala Asn Ser 1 5 10 15 TTC ATT TTTAAG TAT GCA GAT CAG GAT CGG GAG TGC CAC CCT TGC CAT 95 Phe Ile Phe LysTyr Ala Asp Gln Asp Arg Glu Cys His Pro Cys His 20 25 30 CCA AAC TGC ACCCAG GGG TGT AAC GGT CCC ACT AGT CAT GAC TGC ATT 143 Pro Asn Cys Thr GlnGly Cys Asn Gly Pro Thr Ser His Asp Cys Ile 35 40 45 TAC TAC CCA TGG ACGGGC CAT TCC ACT TTA CCA CAA CAC GCT AGA ACT 191 Tyr Tyr Pro Trp Thr GlyHis Ser Thr Leu Pro Gln His Ala Arg Thr 50 55 60 CCA CTG ATT GCA GCC GGAGTC ATT GGA GGC CTC TTC ATC CTG GTG ATC 239 Pro Leu Ile Ala Ala Gly ValIle Gly Gly Leu Phe Ile Leu Val Ile 65 70 75 ATG GCT TTG ACA TTT GCT GTCTAT GTC AGA AGA AAG AGC ATC AAA AAG 287 Met Ala Leu Thr Phe Ala Val TyrVal Arg Arg Lys Ser Ile Lys Lys 80 85 90 95 AAA CGT GCT TTG AGG AGA TTCCTG GAG ACA GAG CTG GTA GAG CCC TTA 335 Lys Arg Ala Leu Arg Arg Phe LeuGlu Thr Glu Leu Val Glu Pro Leu 100 105 110 ACT CCC AGT GGC ACG GCA CCCAAT CAA GCT CAA CTT CGC ATT TTG AAG 383 Thr Pro Ser Gly Thr Ala Pro AsnGln Ala Gln Leu Arg Ile Leu Lys 115 120 125 GAA ACC GAA CTA AAG AGG GTAAAG GTC CTT GGC TCG GGA GCT TTT GGA 431 Glu Thr Glu Leu Lys Arg Val LysVal Leu Gly Ser Gly Ala Phe Gly 130 135 140 ACC GTT TAT AAA GGT ATT TGGGTG CCT GAA GGT GAA ACA GTG AAA ATC 479 Thr Val Tyr Lys Gly Ile Trp ValPro Glu Gly Glu Thr Val Lys Ile 145 150 155 CCT GTG GCT ATA AAG ATC CTCAAT GAA ACA ACT GGC CCC AAA GCC AAC 527 Pro Val Ala Ile Lys Ile Leu AsnGlu Thr Thr Gly Pro Lys Ala Asn 160 165 170 175 GTG GAG TTC ATG GAT GAGGCT CTG ATC ATG GCA AGT ATG GAT CAC CCA 575 Val Glu Phe Met Asp Glu AlaLeu Ile Met Ala Ser Met Asp His Pro 180 185 190 CAC CTA GTT CGC CTA TTGGGA GTG TGT CTG AGT CCC ACT ATC CAG TTG 623 His Leu Val Arg Leu Leu GlyVal Cys Leu Ser Pro Thr Ile Gln Leu 195 200 205 GTT ACG CAG CTG ATG CCGCAT GCG TGC CTA CTG GAC TAT GTT CAT GAA 671 Val Thr Gln Leu Met Pro HisAla Cys Leu Leu Asp Tyr Val His Glu 210 215 220 CAC AAG GAT AAC ATT GGATCA CAG CTG CTG TTG AAC TGG TGT GTC CAG 719 His Lys Asp Asn Ile Gly SerGln Leu Leu Leu Asn Trp Cys Val Gln 225 230 235 ATT GCT AAG GGA ATG ATGTAC CTA GAA GAA AGA CGG CTT GTT CAT CGG 767 Ile Ala Lys Gly Met Met TyrLeu Glu Glu Arg Arg Leu Val His Arg 240 245 250 255 GAT CTG GCA GCC CGCAAT GTC TTA GTG AAA TCT CCA AAT CAT GTT AAA 815 Asp Leu Ala Ala Arg AsnVal Leu Val Lys Ser Pro Asn His Val Lys 260 265 270 ATC ACA GAT TTT GGACTG GCC CGG CTC TTG GAA GGA GAT GAA AAA GAA 863 Ile Thr Asp Phe Gly LeuAla Arg Leu Leu Glu Gly Asp Glu Lys Glu 275 280 285 TAC AAT GCT GAT GGTGGC AAG ATG CCA ATT AAA TGG ATG GCT CTG GAA 911 Tyr Asn Ala Asp Gly GlyLys Met Pro Ile Lys Trp Met Ala Leu Glu 290 295 300 TGT ATA CAT TAT AGGAAA TTC ACA CAT CAA AGT GAT GTT TGG AGC TAT 959 Cys Ile His Tyr Arg LysPhe Thr His Gln Ser Asp Val Trp Ser Tyr 305 310 315 GGC GTC ACT ATA TGGGAA CTG ATG ACC TTT GGA GGA AAG CCC TAT GAT 1007 Gly Val Thr Ile Trp GluLeu Met Thr Phe Gly Gly Lys Pro Tyr Asp 320 325 330 335 GGA ATT CCA ACCCGA GAA ATC CCC GAT TTA CTG GAG AAA GGA GAG CGT 1055 Gly Ile Pro Thr ArgGlu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg 340 345 350 CTG CCT CAG CCTCCC ATC TGC ACT ATT GAT GTT TAC ATG GTC ATG GTC 1103 Leu Pro Gln Pro ProIle Cys Thr Ile Asp Val Tyr Met Val Met Val 355 360 365 AAA TGT TGG ATGATC GAT GCT GAC AGC AGA CCT AAA TTC AAA GAA CTG 1151 Lys Cys Trp Met IleAsp Ala Asp Ser Arg Pro Lys Phe Lys Glu Leu 370 375 380 GCT GCT GAG TTTTCA AGA ATG GCT AGA GAC CCT CAA AGA TAC CTA GTT 1199 Ala Ala Glu Phe SerArg Met Ala Arg Asp Pro Gln Arg Tyr Leu Val 385 390 395 ATT CAG GGT GATGAT CGT ATG AAG CTT CCC AGT CCA AAT GAC AGC AAA 1247 Ile Gln Gly Asp AspArg Met Lys Leu Pro Ser Pro Asn Asp Ser Lys 400 405 410 415 TTC TTC CAGAAT CTC TTG GAT GAA GAG GAT TTG GAA GAC ATG ATG GAT 1295 Phe Phe Gln AsnLeu Leu Asp Glu Glu Asp Leu Glu Asp Met Met Asp 420 425 430 GCT GAG GAATAT TTG GTC CCC CAG GCT TTC AAC ATA CCT CCT CCC ATC 1343 Ala Glu Glu TyrLeu Val Pro Gln Ala Phe Asn Ile Pro Pro Pro Ile 435 440 445 TAC ACA TCCAGA ACA AGA ATT GAC TCC AAT AGG AAT CAG TTT GTG TAC 1391 Tyr Thr Ser ArgThr Arg Ile Asp Ser Asn Arg Asn Gln Phe Val Tyr 450 455 460 CAA GAT GGGGGC TTT GCT ACA CAA CAA GGA ATG CCC ATG CCC TAC AGA 1439 Gln Asp Gly GlyPhe Ala Thr Gln Gln Gly Met Pro Met Pro Tyr Arg 465 470 475 GCC ACA ACCAGC ACC ATA CCA GAG GCT CCA GTA GCT CAG GGT GCA ACG 1487 Ala Thr Thr SerThr Ile Pro Glu Ala Pro Val Ala Gln Gly Ala Thr 480 485 490 495 GCT GAGATG TTT GAT GAC TCC TGC TGT AAT GGT ACC CTA CGA AAG CCA 1535 Ala Glu MetPhe Asp Asp Ser Cys Cys Asn Gly Thr Leu Arg Lys Pro 500 505 510 GTG GCACCC CAT GTC CAA GAG GAC AGT AGC ACT CAG AGG TAT AGT GCT 1583 Val Ala ProHis Val Gln Glu Asp Ser Ser Thr Gln Arg Tyr Ser Ala 515 520 525 GAT CCCACA GTG TTC GCC CCA GAA CGG AAT CCT CGA GGA GAA CTG GAT 1631 Asp Pro ThrVal Phe Ala Pro Glu Arg Asn Pro Arg Gly Glu Leu Asp 530 535 540 GAA GAAGGC TAC ATG ACT CCA ATG CAT GAC AAG CCC AAA CAA GAA TAT 1679 Glu Glu GlyTyr Met Thr Pro Met His Asp Lys Pro Lys Gln Glu Tyr 545 550 555 CTG AATCCT GTG GAA GAG AAC CCT TTT GTG TCC CGA AGG AAG AAT GGA 1727 Leu Asn ProVal Glu Glu Asn Pro Phe Val Ser Arg Arg Lys Asn Gly 560 565 570 575 GATCTT CAA GCT TTA GAT AAT CCG GAG TAT CAC AGT GCT TCC AGC GGT 1775 Asp LeuGln Ala Leu Asp Asn Pro Glu Tyr His Ser Ala Ser Ser Gly 580 585 590 CCACCC AAG GCG GAG GAT GAA TAC GTG AAT GAG CCT CTA TAC CTC AAC 1823 Pro ProLys Ala Glu Asp Glu Tyr Val Asn Glu Pro Leu Tyr Leu Asn 595 600 605 ACCTTC GCC AAT GCC TTG GGG AGT GCA GAG TAC ATG AAA AAC AGT GTA 1871 Thr PheAla Asn Ala Leu Gly Ser Ala Glu Tyr Met Lys Asn Ser Val 610 615 620 CTGTCT GTG CCA GAG AAA GCC AAG AAA GCA TTT GAC AAC CCC GAC TAC 1919 Leu SerVal Pro Glu Lys Ala Lys Lys Ala Phe Asp Asn Pro Asp Tyr 625 630 635 TGGAAC CAC AGC CTG CCA CCC CGG AGC ACC CTT CAG CAC CCA GAC TAC 1967 Trp AsnHis Ser Leu Pro Pro Arg Ser Thr Leu Gln His Pro Asp Tyr 640 645 650 655CTG CAG GAA TAC AGC ACA AAA TAT TTT TAT AAA CAG AAT GGA CGG ATC 2015 LeuGln Glu Tyr Ser Thr Lys Tyr Phe Tyr Lys Gln Asn Gly Arg Ile 660 665 670CGC CCC ATT GTG GCA GAG AAT CCT GAG TAC CTC TCG GAG TTC TCG CTG 2063 ArgPro Ile Val Ala Glu Asn Pro Glu Tyr Leu Ser Glu Phe Ser Leu 675 680 685AAG CCT GGC ACT ATG CTG CCC CCT CCG CCC TAC AGA CAC CGG AAT ACT 2111 LysPro Gly Thr Met Leu Pro Pro Pro Pro Tyr Arg His Arg Asn Thr 690 695 700GTG GTG T GAGCTTGGCT AGAGTGTTAG GTGGAGAAAC ACACACCCAC TCCATTTCCC 2168Val Val 705 TTCCCCCTCC TCTTTCTCTG GTGGTCTTCC TTCTTCTCCC AAGGCCAGTAGTTTTGACAC 2228 TTCCAAGTGG AAGCAGTAGA GATGCAATGA TAGTTCTGTG CTTACCTAACTTGAATATTA 2288 GAAGGAAAGA CTGAAAGAGA AAGACAGGGA TACACACACT GTTTCTTCGTTTCTTCATAT 2348 GGGTTGGTTA ACAGAGTGTC AAAGCTAGAG AAGGTCTAGG AAGTATAAGGCAATACTGCC 2408 TGCTGTCAAA GAGCCCCATC TTTCTTCTC 2437 705 amino acidsamino acid linear protein 4 Asn Cys Val Glu Lys Cys Pro Asp Gly Leu GlnGly Ala Asn Ser Phe 1 5 10 15 Ile Phe Lys Tyr Ala Asp Gln Asp Arg GluCys His Pro Cys His Pro 20 25 30 Asn Cys Thr Gln Gly Cys Asn Gly Pro ThrSer His Asp Cys Ile Tyr 35 40 45 Tyr Pro Trp Thr Gly His Ser Thr Leu ProGln His Ala Arg Thr Pro 50 55 60 Leu Ile Ala Ala Gly Val Ile Gly Gly LeuPhe Ile Leu Val Ile Met 65 70 75 80 Ala Leu Thr Phe Ala Val Tyr Val ArgArg Lys Ser Ile Lys Lys Lys 85 90 95 Arg Ala Leu Arg Arg Phe Leu Glu ThrGlu Leu Val Glu Pro Leu Thr 100 105 110 Pro Ser Gly Thr Ala Pro Asn GlnAla Gln Leu Arg Ile Leu Lys Glu 115 120 125 Thr Glu Leu Lys Arg Val LysVal Leu Gly Ser Gly Ala Phe Gly Thr 130 135 140 Val Tyr Lys Gly Ile TrpVal Pro Glu Gly Glu Thr Val Lys Ile Pro 145 150 155 160 Val Ala Ile LysIle Leu Asn Glu Thr Thr Gly Pro Lys Ala Asn Val 165 170 175 Glu Phe MetAsp Glu Ala Leu Ile Met Ala Ser Met Asp His Pro His 180 185 190 Leu ValArg Leu Leu Gly Val Cys Leu Ser Pro Thr Ile Gln Leu Val 195 200 205 ThrGln Leu Met Pro His Ala Cys Leu Leu Asp Tyr Val His Glu His 210 215 220Lys Asp Asn Ile Gly Ser Gln Leu Leu Leu Asn Trp Cys Val Gln Ile 225 230235 240 Ala Lys Gly Met Met Tyr Leu Glu Glu Arg Arg Leu Val His Arg Asp245 250 255 Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn His Val LysIle 260 265 270 Thr Asp Phe Gly Leu Ala Arg Leu Leu Glu Gly Asp Glu LysGlu Tyr 275 280 285 Asn Ala Asp Gly Gly Lys Met Pro Ile Lys Trp Met AlaLeu Glu Cys 290 295 300 Ile His Tyr Arg Lys Phe Thr His Gln Ser Asp ValTrp Ser Tyr Gly 305 310 315 320 Val Thr Ile Trp Glu Leu Met Thr Phe GlyGly Lys Pro Tyr Asp Gly 325 330 335 Ile Pro Thr Arg Glu Ile Pro Asp LeuLeu Glu Lys Gly Glu Arg Leu 340 345 350 Pro Gln Pro Pro Ile Cys Thr IleAsp Val Tyr Met Val Met Val Lys 355 360 365 Cys Trp Met Ile Asp Ala AspSer Arg Pro Lys Phe Lys Glu Leu Ala 370 375 380 Ala Glu Phe Ser Arg MetAla Arg Asp Pro Gln Arg Tyr Leu Val Ile 385 390 395 400 Gln Gly Asp AspArg Met Lys Leu Pro Ser Pro Asn Asp Ser Lys Phe 405 410 415 Phe Gln AsnLeu Leu Asp Glu Glu Asp Leu Glu Asp Met Met Asp Ala 420 425 430 Glu GluTyr Leu Val Pro Gln Ala Phe Asn Ile Pro Pro Pro Ile Tyr 435 440 445 ThrSer Arg Thr Arg Ile Asp Ser Asn Arg Asn Gln Phe Val Tyr Gln 450 455 460Asp Gly Gly Phe Ala Thr Gln Gln Gly Met Pro Met Pro Tyr Arg Ala 465 470475 480 Thr Thr Ser Thr Ile Pro Glu Ala Pro Val Ala Gln Gly Ala Thr Ala485 490 495 Glu Met Phe Asp Asp Ser Cys Cys Asn Gly Thr Leu Arg Lys ProVal 500 505 510 Ala Pro His Val Gln Glu Asp Ser Ser Thr Gln Arg Tyr SerAla Asp 515 520 525 Pro Thr Val Phe Ala Pro Glu Arg Asn Pro Arg Gly GluLeu Asp Glu 530 535 540 Glu Gly Tyr Met Thr Pro Met His Asp Lys Pro LysGln Glu Tyr Leu 545 550 555 560 Asn Pro Val Glu Glu Asn Pro Phe Val SerArg Arg Lys Asn Gly Asp 565 570 575 Leu Gln Ala Leu Asp Asn Pro Glu TyrHis Ser Ala Ser Ser Gly Pro 580 585 590 Pro Lys Ala Glu Asp Glu Tyr ValAsn Glu Pro Leu Tyr Leu Asn Thr 595 600 605 Phe Ala Asn Ala Leu Gly SerAla Glu Tyr Met Lys Asn Ser Val Leu 610 615 620 Ser Val Pro Glu Lys AlaLys Lys Ala Phe Asp Asn Pro Asp Tyr Trp 625 630 635 640 Asn His Ser LeuPro Pro Arg Ser Thr Leu Gln His Pro Asp Tyr Leu 645 650 655 Gln Glu TyrSer Thr Lys Tyr Phe Tyr Lys Gln Asn Gly Arg Ile Arg 660 665 670 Pro IleVal Ala Glu Asn Pro Glu Tyr Leu Ser Glu Phe Ser Leu Lys 675 680 685 ProGly Thr Met Leu Pro Pro Pro Pro Tyr Arg His Arg Asn Thr Val 690 695 700Val 705 3307 base pairs nucleic acid single linear DNA Tyro-3 CDS237..2859 5 CGGCGGCGGC GGCGGCTGTG GAAGGAGCGC GGTGGCCCAG CCGCAGCCCCGGGGACTCCT 60 CGCTGCTGAC GGCGGTGGCC GCGGCTCTAG GCGGCCGCGG GTCCGGGACGCCCGGGCCGA 120 GCGCCGCCCC CCGCCCCTCC CGCGGGCCTC CCGCCCCTCC TCCGCCACCCTCCTCTCTGC 180 GCTCGCGGGC CGGGCCCGGC ATGGTGCGGC GTCGCCGCCG ATGGCTGAGGCGGAGC 236 ATG GGG TGG CCG GGG CTC CGG CCG CTG CTG CTG GCG GGA CTG GCTTCT 284 Met Gly Trp Pro Gly Leu Arg Pro Leu Leu Leu Ala Gly Leu Ala Ser1 5 10 15 CTG CTG CTC CCC GGG TCT GCG GCC GCA GGC CTG AAG CTC ATG GGCGCC 332 Leu Leu Leu Pro Gly Ser Ala Ala Ala Gly Leu Lys Leu Met Gly Ala20 25 30 CCA GTG AAG ATG ACC GTG TCT CAG GGG CAG CCA GTG AAG CTC AAC TGC380 Pro Val Lys Met Thr Val Ser Gln Gly Gln Pro Val Lys Leu Asn Cys 3540 45 AGC GTG GAG GGG ATG GAG GAC CCT GAC ATC CAC TGG ATG AAG GAT GGC428 Ser Val Glu Gly Met Glu Asp Pro Asp Ile His Trp Met Lys Asp Gly 5055 60 ACC GTG GTC CAG AAT GCA AGC CAG GTG TCC ATC TCC ATC AGC GAG CAC476 Thr Val Val Gln Asn Ala Ser Gln Val Ser Ile Ser Ile Ser Glu His 6570 75 80 AGC TGG ATT GGC TTA CTC AGC CTA AAG TCA GTG GAA CGG TCT GAT GCT524 Ser Trp Ile Gly Leu Leu Ser Leu Lys Ser Val Glu Arg Ser Asp Ala 8590 95 GGC CTG TAC TGG TGC CAG GTG AAG GAT GGG GAG GAA ACC AAG ATT TCT572 Gly Leu Tyr Trp Cys Gln Val Lys Asp Gly Glu Glu Thr Lys Ile Ser 100105 110 CAG TCA GTA TGG CTC ACT GTC GAA GGT GTG CCA TTC TTC ACA GTG GAA620 Gln Ser Val Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu 115120 125 CCA AAA GAT CTG GCG GTG CCA CCC AAT GCC CCT TTT CAG CTG TCT TGT668 Pro Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gln Leu Ser Cys 130135 140 GAG GCT GTG GGT CCT CCA GAA CCC GTA ACC ATT TAC TGG TGG AGA GGA716 Glu Ala Val Gly Pro Pro Glu Pro Val Thr Ile Tyr Trp Trp Arg Gly 145150 155 160 CTC ACT AAG GTT GGG GGA CCT GCT CCC TCT CCC TCT GTT TTA AATGTG 764 Leu Thr Lys Val Gly Gly Pro Ala Pro Ser Pro Ser Val Leu Asn Val165 170 175 ACA GGA GTG ACC CAG CGC ACA GAG TTT TCT TGT GAA GCC CGC AACATA 812 Thr Gly Val Thr Gln Arg Thr Glu Phe Ser Cys Glu Ala Arg Asn Ile180 185 190 AAA GGC CTG GCC ACT TCC CGA CCA GCC ATT GTT CGC CTT CAA GCACCG 860 Lys Gly Leu Ala Thr Ser Arg Pro Ala Ile Val Arg Leu Gln Ala Pro195 200 205 CCT GCA GCT CCT TTC AAC ACC ACA GTA ACA ACG ATC TCC AGC TACAAC 908 Pro Ala Ala Pro Phe Asn Thr Thr Val Thr Thr Ile Ser Ser Tyr Asn210 215 220 GCT AGC GTG GCC TGG GTG CCA GGT GCT GAC GGC CTA GCT CTG CTGCAT 956 Ala Ser Val Ala Trp Val Pro Gly Ala Asp Gly Leu Ala Leu Leu His225 230 235 240 TCC TGT ACT GTA CAG GTG GCA CAC GCC CCA GGA GAA TGG GAGGCC CTT 1004 Ser Cys Thr Val Gln Val Ala His Ala Pro Gly Glu Trp Glu AlaLeu 245 250 255 GCT GTT GTG GTT CCT GTG CCA CCT TTT ACC TGC CTG CTT CGGAAC TTG 1052 Ala Val Val Val Pro Val Pro Pro Phe Thr Cys Leu Leu Arg AsnLeu 260 265 270 GCC CCT GCC ACC AAC TAC AGC CTT AGG GTG CGC TGT GCC AATGCC TTG 1100 Ala Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys Ala Asn AlaLeu 275 280 285 GGC CCT TCT CCC TAC GGC GAC TGG GTG CCC TTT CAG ACA AAGGGC CTA 1148 Gly Pro Ser Pro Tyr Gly Asp Trp Val Pro Phe Gln Thr Lys GlyLeu 290 295 300 GCG CCA CGC AGA GCT CCT CAG AAT TTC CAT GCC ATT CGT ACCGAC TCA 1196 Ala Pro Arg Arg Ala Pro Gln Asn Phe His Ala Ile Arg Thr AspSer 305 310 315 320 GGC CTT ATC CTG GAA TGG GAA GAA GTG ATT CCT GAG GACCCT GGG GAA 1244 Gly Leu Ile Leu Glu Trp Glu Glu Val Ile Pro Glu Asp ProGly Glu 325 330 335 GGC CCC CTA GGA CCT TAT AAG CTG TCC TGG GTC CAA GAAAAT GGA ACC 1292 Gly Pro Leu Gly Pro Tyr Lys Leu Ser Trp Val Gln Glu AsnGly Thr 340 345 350 CAG GAT GAG CTG ATG GTG GAA GGG ACC AGG GCC AAT CTGACC GAC TGG 1340 Gln Asp Glu Leu Met Val Glu Gly Thr Arg Ala Asn Leu ThrAsp Trp 355 360 365 GTA CCC CAG AAG GAC CTG ATT TTG CGT GTG TGT GCC TCCAAT GCA ATT 1388 Val Pro Gln Lys Asp Leu Ile Leu Arg Val Cys Ala Ser AsnAla Ile 370 375 380 GGT GAT GGG CCC TGG AGT CAG CCA CTG GTG GTG TCT TCTCAT GAC CAT 1436 Gly Asp Gly Pro Trp Ser Gln Pro Leu Val Val Ser Ser HisAsp His 385 390 395 400 GCA GGG AGG CAG GGC CCT CCC CAC AGC CGC ACA TCCTGG GTG CCT GTG 1484 Ala Gly Arg Gln Gly Pro Pro His Ser Arg Thr Ser TrpVal Pro Val 405 410 415 GTC CTG GGC GTG CTC ACC GCC CTG ATC ACA GCT GCTGCC TTG GCC CTC 1532 Val Leu Gly Val Leu Thr Ala Leu Ile Thr Ala Ala AlaLeu Ala Leu 420 425 430 ATC CTG CTT CGG AAG AGA CGC AAG GAG ACG CGT TTCGGG CAA GCC TTT 1580 Ile Leu Leu Arg Lys Arg Arg Lys Glu Thr Arg Phe GlyGln Ala Phe 435 440 445 GAC AGT GTC ATG GCC CGA GGG GAG CCA GCT GTA CACTTC CGG GCA GCC 1628 Asp Ser Val Met Ala Arg Gly Glu Pro Ala Val His PheArg Ala Ala 450 455 460 CGA TCT TTC AAT CGA GAA AGG CCT GAA CGC ATT GAGGCC ACA TTG GAT 1676 Arg Ser Phe Asn Arg Glu Arg Pro Glu Arg Ile Glu AlaThr Leu Asp 465 470 475 480 AGC CTG GGC ATC AGC GAT GAA TTG AAG GAA AAGCTG GAG GAT GTC CTC 1724 Ser Leu Gly Ile Ser Asp Glu Leu Lys Glu Lys LeuGlu Asp Val Leu 485 490 495 ATT CCA GAG CAG CAG TTC ACC CTC GGT CGG ATGTTG GGC AAA GGA GAG 1772 Ile Pro Glu Gln Gln Phe Thr Leu Gly Arg Met LeuGly Lys Gly Glu 500 505 510 TTT GGA TCA GTG CGG GAA GCC CAG CTA AAG CAGGAA GAT GGC TCC TTC 1820 Phe Gly Ser Val Arg Glu Ala Gln Leu Lys Gln GluAsp Gly Ser Phe 515 520 525 GTG AAA GTG GCA GTG AAG ATG CTG AAA GCT GACATC ATT GCC TCA AGC 1868 Val Lys Val Ala Val Lys Met Leu Lys Ala Asp IleIle Ala Ser Ser 530 535 540 GAC ATA GAA GAG TTC CTC CGG GAA GCA GCT TGCATG AAG GAG TTT GAC 1916 Asp Ile Glu Glu Phe Leu Arg Glu Ala Ala Cys MetLys Glu Phe Asp 545 550 555 560 CAT CCA CAC GTG GCC AAG CTT GTT GGG GTGAGC CTC CGG AGC AGG GCT 1964 His Pro His Val Ala Lys Leu Val Gly Val SerLeu Arg Ser Arg Ala 565 570 575 AAA GGT CGT CTC CCC ATT CCC ATG GTC ATCCTG CCC TTC ATG AAA CAT 2012 Lys Gly Arg Leu Pro Ile Pro Met Val Ile LeuPro Phe Met Lys His 580 585 590 GGA GAC TTG CAC GCC TTT CTG CTC GCC TCCCGA ATC GGG GAG AAC CCT 2060 Gly Asp Leu His Ala Phe Leu Leu Ala Ser ArgIle Gly Glu Asn Pro 595 600 605 TTT AAC CTG CCC CTC CAG ACC CTG GTC CGGTTC ATG GTG GAC ATT CGC 2108 Phe Asn Leu Pro Leu Gln Thr Leu Val Arg PheMet Val Asp Ile Arg 610 615 620 TGT GGC ATG GAG TAC CTG AGC TCC CGG AACTTC ATC CAC CGA GAC CTA 2156 Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn PheIle His Arg Asp Leu 625 630 635 640 GCA GCT CGG AAT TGC ATG CTG GCC GAGGAC ATG ACA GTG TGT GTG GCT 2204 Ala Ala Arg Asn Cys Met Leu Ala Glu AspMet Thr Val Cys Val Ala 645 650 655 GAT TTT GGA CTC TCT CGG AAA ATC TATAGC GGG GAC TAT TAT CGT CAG 2252 Asp Phe Gly Leu Ser Arg Lys Ile Tyr SerGly Asp Tyr Tyr Arg Gln 660 665 670 GGC TGT GCC TCC AAA TTG CCC GTC AAGTGG CTG GCC CTG GAG AGC TTG 2300 Gly Cys Ala Ser Lys Leu Pro Val Lys TrpLeu Ala Leu Glu Ser Leu 675 680 685 GCT GAC AAC TTG TAT ACT GTA CAC AGTGAT GTG TGG GCC TTC GGG GTG 2348 Ala Asp Asn Leu Tyr Thr Val His Ser AspVal Trp Ala Phe Gly Val 690 695 700 ACC ATG TGG GAG ATC ATG ACT CGT GGGCAG ACG CCA TAT GCT GGC ATT 2396 Thr Met Trp Glu Ile Met Thr Arg Gly GlnThr Pro Tyr Ala Gly Ile 705 710 715 720 GAA AAT GCC GAG ATT TAC AAC TACCTC ATC GGC GGG AAC CGC CTG AAG 2444 Glu Asn Ala Glu Ile Tyr Asn Tyr LeuIle Gly Gly Asn Arg Leu Lys 725 730 735 CAG CCT CCG GAG TGC ATG GAG GAAGTG TAT GAT CTC ATG TAC CAG TGC 2492 Gln Pro Pro Glu Cys Met Glu Glu ValTyr Asp Leu Met Tyr Gln Cys 740 745 750 TGG AGC GCC GAC CCC AAG CAG CGCCCA AGC TTC ACG TGT CTG CGA ATG 2540 Trp Ser Ala Asp Pro Lys Gln Arg ProSer Phe Thr Cys Leu Arg Met 755 760 765 GAA CTG GAG AAC ATT CTG GGC CACCTG TCT GTG CTG TCC ACC AGC CAG 2588 Glu Leu Glu Asn Ile Leu Gly His LeuSer Val Leu Ser Thr Ser Gln 770 775 780 GAC CCC TTG TAC ATC AAC ATT GAGAGA GCT GAG CAG CCT ACT GAG AGT 2636 Asp Pro Leu Tyr Ile Asn Ile Glu ArgAla Glu Gln Pro Thr Glu Ser 785 790 795 800 GGC AGC CCT GAG GTC CAC TGTGGA GAG CGA TCC AGC AGC GAG GCA GGG 2684 Gly Ser Pro Glu Val His Cys GlyGlu Arg Ser Ser Ser Glu Ala Gly 805 810 815 GAC GGC AGT GGC GTG GGG GCAGTA GGT GGC ATC CCC AGT GAC TCT CGG 2732 Asp Gly Ser Gly Val Gly Ala ValGly Gly Ile Pro Ser Asp Ser Arg 820 825 830 TAC ATC TTC AGC CCC GGA GGGCTA TCC GAG TCA CCA GGG CAG CTG GAG 2780 Tyr Ile Phe Ser Pro Gly Gly LeuSer Glu Ser Pro Gly Gln Leu Glu 835 840 845 CAG CAG CCA GAA AGC CCC CTCAAT GAG AAC CAG AGG CTG TTG TTG CTG 2828 Gln Gln Pro Glu Ser Pro Leu AsnGlu Asn Gln Arg Leu Leu Leu Leu 850 855 860 CAG CAA GGG CTA CTG CCT CACAGT AGC TGT T AACCCTCAGG CAGAGGAAAG 2879 Gln Gln Gly Leu Leu Pro His SerSer Cys 865 870 TTGGGGCCCC TGGCTCTGCT GACCGCTGTG CTGCCTGACT AGGCCCAGTCTGATCACAGC 2939 CCAGGCAGCA AGGTATGGAG GCTCCTGTGG TAGCCCTCCC AAGCTGTGCTGGCGCCTGGA 2999 CGGACCAAAT TGCCCAATCC CAGTTCTTCC TGCAGCCGCT CTGGCCAGCCTGGCATCAGT 3059 TCAGGCCTTG GCTTACAGGA GGTGAGCCAG AGCTGGTTGC CTGAATGCAGGCAGCTGGCA 3119 GGAGGGGAGG GTGGCTATGT TTCCATGGGT ACCATGGTTG TGGATGGCAGTAAGGGAGGG 3179 TAGCAACAGC CCTGTGCGCC CTACCCTCCT GGCTGAGCTG CTCCTACTTTAGTGCATGCT 3239 TGGAGCCGCC TGCAGCCTGG AACTCAGCAC TGCCCACCAC ACTTGGGCCGAAATGCCAGG 3299 TTTGCCCC 3307 874 amino acids amino acid linear protein6 Met Gly Trp Pro Gly Leu Arg Pro Leu Leu Leu Ala Gly Leu Ala Ser 1 5 1015 Leu Leu Leu Pro Gly Ser Ala Ala Ala Gly Leu Lys Leu Met Gly Ala 20 2530 Pro Val Lys Met Thr Val Ser Gln Gly Gln Pro Val Lys Leu Asn Cys 35 4045 Ser Val Glu Gly Met Glu Asp Pro Asp Ile His Trp Met Lys Asp Gly 50 5560 Thr Val Val Gln Asn Ala Ser Gln Val Ser Ile Ser Ile Ser Glu His 65 7075 80 Ser Trp Ile Gly Leu Leu Ser Leu Lys Ser Val Glu Arg Ser Asp Ala 8590 95 Gly Leu Tyr Trp Cys Gln Val Lys Asp Gly Glu Glu Thr Lys Ile Ser100 105 110 Gln Ser Val Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr ValGlu 115 120 125 Pro Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gln LeuSer Cys 130 135 140 Glu Ala Val Gly Pro Pro Glu Pro Val Thr Ile Tyr TrpTrp Arg Gly 145 150 155 160 Leu Thr Lys Val Gly Gly Pro Ala Pro Ser ProSer Val Leu Asn Val 165 170 175 Thr Gly Val Thr Gln Arg Thr Glu Phe SerCys Glu Ala Arg Asn Ile 180 185 190 Lys Gly Leu Ala Thr Ser Arg Pro AlaIle Val Arg Leu Gln Ala Pro 195 200 205 Pro Ala Ala Pro Phe Asn Thr ThrVal Thr Thr Ile Ser Ser Tyr Asn 210 215 220 Ala Ser Val Ala Trp Val ProGly Ala Asp Gly Leu Ala Leu Leu His 225 230 235 240 Ser Cys Thr Val GlnVal Ala His Ala Pro Gly Glu Trp Glu Ala Leu 245 250 255 Ala Val Val ValPro Val Pro Pro Phe Thr Cys Leu Leu Arg Asn Leu 260 265 270 Ala Pro AlaThr Asn Tyr Ser Leu Arg Val Arg Cys Ala Asn Ala Leu 275 280 285 Gly ProSer Pro Tyr Gly Asp Trp Val Pro Phe Gln Thr Lys Gly Leu 290 295 300 AlaPro Arg Arg Ala Pro Gln Asn Phe His Ala Ile Arg Thr Asp Ser 305 310 315320 Gly Leu Ile Leu Glu Trp Glu Glu Val Ile Pro Glu Asp Pro Gly Glu 325330 335 Gly Pro Leu Gly Pro Tyr Lys Leu Ser Trp Val Gln Glu Asn Gly Thr340 345 350 Gln Asp Glu Leu Met Val Glu Gly Thr Arg Ala Asn Leu Thr AspTrp 355 360 365 Val Pro Gln Lys Asp Leu Ile Leu Arg Val Cys Ala Ser AsnAla Ile 370 375 380 Gly Asp Gly Pro Trp Ser Gln Pro Leu Val Val Ser SerHis Asp His 385 390 395 400 Ala Gly Arg Gln Gly Pro Pro His Ser Arg ThrSer Trp Val Pro Val 405 410 415 Val Leu Gly Val Leu Thr Ala Leu Ile ThrAla Ala Ala Leu Ala Leu 420 425 430 Ile Leu Leu Arg Lys Arg Arg Lys GluThr Arg Phe Gly Gln Ala Phe 435 440 445 Asp Ser Val Met Ala Arg Gly GluPro Ala Val His Phe Arg Ala Ala 450 455 460 Arg Ser Phe Asn Arg Glu ArgPro Glu Arg Ile Glu Ala Thr Leu Asp 465 470 475 480 Ser Leu Gly Ile SerAsp Glu Leu Lys Glu Lys Leu Glu Asp Val Leu 485 490 495 Ile Pro Glu GlnGln Phe Thr Leu Gly Arg Met Leu Gly Lys Gly Glu 500 505 510 Phe Gly SerVal Arg Glu Ala Gln Leu Lys Gln Glu Asp Gly Ser Phe 515 520 525 Val LysVal Ala Val Lys Met Leu Lys Ala Asp Ile Ile Ala Ser Ser 530 535 540 AspIle Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys Glu Phe Asp 545 550 555560 His Pro His Val Ala Lys Leu Val Gly Val Ser Leu Arg Ser Arg Ala 565570 575 Lys Gly Arg Leu Pro Ile Pro Met Val Ile Leu Pro Phe Met Lys His580 585 590 Gly Asp Leu His Ala Phe Leu Leu Ala Ser Arg Ile Gly Glu AsnPro 595 600 605 Phe Asn Leu Pro Leu Gln Thr Leu Val Arg Phe Met Val AspIle Arg 610 615 620 Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn Phe Ile HisArg Asp Leu 625 630 635 640 Ala Ala Arg Asn Cys Met Leu Ala Glu Asp MetThr Val Cys Val Ala 645 650 655 Asp Phe Gly Leu Ser Arg Lys Ile Tyr SerGly Asp Tyr Tyr Arg Gln 660 665 670 Gly Cys Ala Ser Lys Leu Pro Val LysTrp Leu Ala Leu Glu Ser Leu 675 680 685 Ala Asp Asn Leu Tyr Thr Val HisSer Asp Val Trp Ala Phe Gly Val 690 695 700 Thr Met Trp Glu Ile Met ThrArg Gly Gln Thr Pro Tyr Ala Gly Ile 705 710 715 720 Glu Asn Ala Glu IleTyr Asn Tyr Leu Ile Gly Gly Asn Arg Leu Lys 725 730 735 Gln Pro Pro GluCys Met Glu Glu Val Tyr Asp Leu Met Tyr Gln Cys 740 745 750 Trp Ser AlaAsp Pro Lys Gln Arg Pro Ser Phe Thr Cys Leu Arg Met 755 760 765 Glu LeuGlu Asn Ile Leu Gly His Leu Ser Val Leu Ser Thr Ser Gln 770 775 780 AspPro Leu Tyr Ile Asn Ile Glu Arg Ala Glu Gln Pro Thr Glu Ser 785 790 795800 Gly Ser Pro Glu Val His Cys Gly Glu Arg Ser Ser Ser Glu Ala Gly 805810 815 Asp Gly Ser Gly Val Gly Ala Val Gly Gly Ile Pro Ser Asp Ser Arg820 825 830 Tyr Ile Phe Ser Pro Gly Gly Leu Ser Glu Ser Pro Gly Gln LeuGlu 835 840 845 Gln Gln Pro Glu Ser Pro Leu Asn Glu Asn Gln Arg Leu LeuLeu Leu 850 855 860 Gln Gln Gly Leu Leu Pro His Ser Ser Cys 865 870 165base pairs nucleic acid single linear DNA Tyro-4 CDS 1..165 7 AAC ATCTTG ATC AAC AGT AAC TTG GTG TGC AAA GTC TCT GAC TTC GGA 48 Asn Ile LeuIle Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 CTT TCTCGA GTG TTG GAA GAT GAC CCT GAA GCT GCT TAC ACC ACC AGA 96 Leu Ser ArgVal Leu Glu Asp Asp Pro Glu Ala Ala Tyr Thr Thr Arg 20 25 30 GGA GGA AAGATA CCA ATA AGG TGG ACA TCA CCA GAA GCA ATT GCC TAC 144 Gly Gly Lys IlePro Ile Arg Trp Thr Ser Pro Glu Ala Ile Ala Tyr 35 40 45 CGC AAG TTC ACATCA GCC AGC 165 Arg Lys Phe Thr Ser Ala Ser 50 55 55 amino acids aminoacid linear protein 8 Asn Ile Leu Ile Asn Ser Asn Leu Val Cys Lys ValSer Asp Phe Gly 1 5 10 15 Leu Ser Arg Val Leu Glu Asp Asp Pro Glu AlaAla Tyr Thr Thr Arg 20 25 30 Gly Gly Lys Ile Pro Ile Arg Trp Thr Ser ProGlu Ala Ile Ala Tyr 35 40 45 Arg Lys Phe Thr Ser Ala Ser 50 55 171 basepairs nucleic acid single linear DNA Tyro-5 CDS 1..171 9 AAC ATC CTT GTCAAT AGC AAC CTG GTG TGC AAG GTG TCT GAC TTC GGG 48 Asn Ile Leu Val AsnSer Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 CTC TCA CGC TTCCTG GAG GAC GAC ACA TCT GAC CCC ACC TAC ACC AGC 96 Leu Ser Arg Phe LeuGlu Asp Asp Thr Ser Asp Pro Thr Tyr Thr Ser 20 25 30 GCT CTG GGT GGG AAGATC CCC ATC CGT TGG ACA GCA CCG GAA GCC ATC 144 Ala Leu Gly Gly Lys IlePro Ile Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 CAG TAC CGG AAA TTC ACCTCA GCC AGT 171 Gln Tyr Arg Lys Phe Thr Ser Ala Ser 50 55 57 amino acidsamino acid linear protein 10 Asn Ile Leu Val Asn Ser Asn Leu Val Cys LysVal Ser Asp Phe Gly 1 5 10 15 Leu Ser Arg Phe Leu Glu Asp Asp Thr SerAsp Pro Thr Tyr Thr Ser 20 25 30 Ala Leu Gly Gly Lys Ile Pro Ile Arg TrpThr Ala Pro Glu Ala Ile 35 40 45 Gln Tyr Arg Lys Phe Thr Ser Ala Ser 5055 171 base pairs nucleic acid single linear DNA Tyro-6 CDS 1..171 11AAC ATC CTT GTC AAC AGT AAC TTG GTC TGC AAA GTA TCT GAC TTT GGG 48 AsnIle Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15CTC TCC CGC TTC CTG GAG GAC GAC CCC TCA GAC CCC ACC TAC ACC AGC 96 LeuSer Arg Phe Leu Glu Asp Asp Pro Ser Asp Pro Thr Tyr Thr Ser 20 25 30 TCCCTG GGT GGG AAG ATC CCT ATC CGT TGG ACC GCC CCA GAG GCC ATA 144 Ser LeuGly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 GCC TATCGG AAG TTC ACG TCT GCC AGC 171 Ala Tyr Arg Lys Phe Thr Ser Ala Ser 5055 57 amino acids amino acid linear protein 12 Asn Ile Leu Val Asn SerAsn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 Leu Ser Arg Phe LeuGlu Asp Asp Pro Ser Asp Pro Thr Tyr Thr Ser 20 25 30 Ser Leu Gly Gly LysIle Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 Ala Tyr Arg Lys PheThr Ser Ala Ser 50 55 162 base pairs nucleic acid single linear DNATyro-7 CDS 1..162 13 AAC TGC ATG CTG AAT GAG AAC ATG TCC GTG TGC GTG GCAGAC TTC GGG 48 Asn Cys Met Leu Asn Glu Asn Met Ser Val Cys Val Ala AspPhe Gly 1 5 10 15 CTC TCC AAG AAG ATC TAC AAT GGG GAT TAC TAC CGC CAAGGG CGC ATT 96 Leu Ser Lys Lys Ile Tyr Asn Gly Asp Tyr Tyr Arg Gln GlyArg Ile 20 25 30 GCC AAG ATG CCA GTC AAG TGG ATT GCT ATC GAG AGT CTG GCAGAT CGA 144 Ala Lys Met Pro Val Lys Trp Ile Ala Ile Glu Ser Leu Ala AspArg 35 40 45 GTC TAC ACC AGC AAG AGT 162 Val Tyr Thr Ser Lys Ser 50 54amino acids amino acid linear protein 14 Asn Cys Met Leu Asn Glu Asn MetSer Val Cys Val Ala Asp Phe Gly 1 5 10 15 Leu Ser Lys Lys Ile Tyr AsnGly Asp Tyr Tyr Arg Gln Gly Arg Ile 20 25 30 Ala Lys Met Pro Val Lys TrpIle Ala Ile Glu Ser Leu Ala Asp Arg 35 40 45 Val Tyr Thr Ser Lys Ser 50159 base pairs nucleic acid single linear DNA Tyro-8 CDS 1..159 15 AACTGT TTG GTG GAC AGT GAT CTC TCC GTG AAA GTC TCA GAC TTT GGA 48 Asn CysLeu Val Asp Ser Asp Leu Ser Val Lys Val Ser Asp Phe Gly 1 5 10 15 ATGACG AGA TAT GTC CTT GAT GAC CAG TAT GTC AGT TCA GTA GGA ACC 96 Met ThrArg Tyr Val Leu Asp Asp Gln Tyr Val Ser Ser Val Gly Thr 20 25 30 AAG TTTCCA GTC AAG TGG TCG GCC CCA GAG GTG TTT CAC TAT TTC AAA 144 Lys Phe ProVal Lys Trp Ser Ala Pro Glu Val Phe His Tyr Phe Lys 35 40 45 TAC AGC AGCAAG TCG 159 Tyr Ser Ser Lys Ser 50 53 amino acids amino acid linearprotein 16 Asn Cys Leu Val Asp Ser Asp Leu Ser Val Lys Val Ser Asp PheGly 1 5 10 15 Met Thr Arg Tyr Val Leu Asp Asp Gln Tyr Val Ser Ser ValGly Thr 20 25 30 Lys Phe Pro Val Lys Trp Ser Ala Pro Glu Val Phe His TyrPhe Lys 35 40 45 Tyr Ser Ser Lys Ser 50 162 base pairs nucleic acidsingle linear DNA Tyro-9 CDS 1..162 17 AAC GTG CTG GTG ACC GAG GAT GACGTG ATG AAG ATC GCT GAC TTT GGT 48 Asn Val Leu Val Thr Glu Asp Asp ValMet Lys Ile Ala Asp Phe Gly 1 5 10 15 CTG GCC CGT GGT GTC CAC CAC ATCGAC TAC TAT AAG AAA ACC AGC AAT 96 Leu Ala Arg Gly Val His His Ile AspTyr Tyr Lys Lys Thr Ser Asn 20 25 30 GGC CGC CTG CCA GTC AAG TGG ATG GCTCCT GAG GCG TTG TTT GAC CGT 144 Gly Arg Leu Pro Val Lys Trp Met Ala ProGlu Ala Leu Phe Asp Arg 35 40 45 GTA TAC ACA CAC CAG AGT 162 Val Tyr ThrHis Gln Ser 50 54 amino acids amino acid linear protein 18 Asn Val LeuVal Thr Glu Asp Asp Val Met Lys Ile Ala Asp Phe Gly 1 5 10 15 Leu AlaArg Gly Val His His Ile Asp Tyr Tyr Lys Lys Thr Ser Asn 20 25 30 Gly ArgLeu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg 35 40 45 Val TyrThr His Gln Ser 50 3120 base pairs nucleic acid single linear DNATyro-10 CDS 485..3047 19 GGGCCCGGGT CTAAGTGGAC TTCTCTTGGT GTGTCAGGAAAAGTTCGGAA AAGCGGCAGA 60 GGGCAGAGTT TGAATCAGGG CGGAAGGGCA GGGAGCTGGGCTCTTCAAGA CTCAGGACCG 120 AGGCAGATCT CATGTTTTGG GGTCTGGATT TGTGTCAGCGAGGGAAGAAC AGGCGCCAAT 180 AACCAAAGAA GGCTGAAGCG AGGTACAGGA CTCCATAGCAGCTGCAAGTA CAATAAACAG 240 TTTTAGCAGA GCTGGAAATG TTGGCAGGCA AGACAGGCCGATCGCAGAGT CGGGCTGCTG 300 GAGAGAGGGA AATCTACAAG CGACCTGACA TTTGGTGCTCTAGAGCATTC TAAGGCTTGC 360 TGCTTGACTT CTAAAGAAGC TGAAATAATT GAGGAGGAGCGGGGACCCTC TGTTTCCAAG 420 GACTCTGTTC TGCAGAGAAT GTTCTGCACC CTCTGATACTCCAGATCCAA CTCCGTCTTC 480 TGAA ATG ATC CCG ATT CCC AGA ATG CCC CTG GTGCTG CTC CTG CTC TTG 529 Met Ile Pro Ile Pro Arg Met Pro Leu Val Leu LeuLeu Leu Leu 1 5 10 15 CTC ATC CTG GGT TCT GCA AAA GCT CAG GTT AAT CCAGCC ATA TGC CGC 577 Leu Ile Leu Gly Ser Ala Lys Ala Gln Val Asn Pro AlaIle Cys Arg 20 25 30 TAT CCT CTG GGC ATG TCA GGA GGC CAC ATT CCA GAT GAGGAC ATC ACA 625 Tyr Pro Leu Gly Met Ser Gly Gly His Ile Pro Asp Glu AspIle Thr 35 40 45 GCC TCA AGT CAG TGG TCA GAA TCC ACG GCT GCC AAA TAT GGGAGG CTG 673 Ala Ser Ser Gln Trp Ser Glu Ser Thr Ala Ala Lys Tyr Gly ArgLeu 50 55 60 GAC TCT GAA GAA GGA GAT GGA GCC TGG TGT CCT GAG ATT CCA GTGCAA 721 Asp Ser Glu Glu Gly Asp Gly Ala Trp Cys Pro Glu Ile Pro Val Gln65 70 75 CCC GAT GAC CTG AAG GAA TTT CTG CAG ATT GAC TTG CGA ACC CTA CAC769 Pro Asp Asp Leu Lys Glu Phe Leu Gln Ile Asp Leu Arg Thr Leu His 8085 90 95 TTT ATC ACT CTT GTG GGG ACC CAG GGG CGC CAT GCA GGG GGT CAT GGC817 Phe Ile Thr Leu Val Gly Thr Gln Gly Arg His Ala Gly Gly His Gly 100105 110 ATT GAA TTT GCA CCC ATG TAC AAG ATC AAC TAC AGT CGG GAT GGC AGT865 Ile Glu Phe Ala Pro Met Tyr Lys Ile Asn Tyr Ser Arg Asp Gly Ser 115120 125 CGC TGG ATC TCC TGG CGT AAC CGG CAT GGG AAG CAG GTG CTT GAT GGA913 Arg Trp Ile Ser Trp Arg Asn Arg His Gly Lys Gln Val Leu Asp Gly 130135 140 AAC AGT AAC CCT TAT GAT GTA TTC CTG AAG GAC TTG GAG CCA CCC ATC961 Asn Ser Asn Pro Tyr Asp Val Phe Leu Lys Asp Leu Glu Pro Pro Ile 145150 155 GTC GCC AGA TTT GTT CGC CTT ATC CCA GTC ACT GAC CAC TCC ATG AAC1009 Val Ala Arg Phe Val Arg Leu Ile Pro Val Thr Asp His Ser Met Asn 160165 170 175 GTG TGC ATG AGG GTT GAG CTT TAT GGT TGT GTC TGG CTA GAT GGCTTG 1057 Val Cys Met Arg Val Glu Leu Tyr Gly Cys Val Trp Leu Asp Gly Leu180 185 190 GTA TCC TAC AAT GCT CCA GCT GGA CAG CAG TTT GTA CTC CCT GGAGGC 1105 Val Ser Tyr Asn Ala Pro Ala Gly Gln Gln Phe Val Leu Pro Gly Gly195 200 205 TCC ATC ATT TAT CTG AAT GAT TCT GTC TAT GAT GGA GCT GTT GGGTAC 1153 Ser Ile Ile Tyr Leu Asn Asp Ser Val Tyr Asp Gly Ala Val Gly Tyr210 215 220 AGC ATG ACT GAA GGG CTA GGC CAG TTG ACT GAT GGA GTA TCC GGCCTG 1201 Ser Met Thr Glu Gly Leu Gly Gln Leu Thr Asp Gly Val Ser Gly Leu225 230 235 GAT GAT TTT ACC CAG ACC CAT GAA TAC CAC GTG TGG CCT GGC TATGAC 1249 Asp Asp Phe Thr Gln Thr His Glu Tyr His Val Trp Pro Gly Tyr Asp240 245 250 255 TAC GTG GGA TGG CGG AAC GAA AGT GCT ACC AAC GGT TTC ATTGAG ATC 1297 Tyr Val Gly Trp Arg Asn Glu Ser Ala Thr Asn Gly Phe Ile GluIle 260 265 270 ATG TTT GAA TTT GAC CGA ATC AGG AAT TTT ACT ACC ATG AAGGTC CAC 1345 Met Phe Glu Phe Asp Arg Ile Arg Asn Phe Thr Thr Met Lys ValHis 275 280 285 TGC AAC AAC ATG TTT GCT AAA GGT GTG AAG ATT TTT AAG GAGGTC CAG 1393 Cys Asn Asn Met Phe Ala Lys Gly Val Lys Ile Phe Lys Glu ValGln 290 295 300 TGC TAC TTT CGC TCG GAA GCC AGC GAG TGG GAA CCC ACT GCTGTC TAC 1441 Cys Tyr Phe Arg Ser Glu Ala Ser Glu Trp Glu Pro Thr Ala ValTyr 305 310 315 TTT CCC CTG GTC CTG GAC GAT GTG AAC CCC AGT GCC CGG TTTGTC ACG 1489 Phe Pro Leu Val Leu Asp Asp Val Asn Pro Ser Ala Arg Phe ValThr 320 325 330 335 GTG CCC CTC CAC CAC CGA ATG GCC AGT GCC ATC AAG TGCCAA TAC CAT 1537 Val Pro Leu His His Arg Met Ala Ser Ala Ile Lys Cys GlnTyr His 340 345 350 TTT GCC GAC ACG TGG ATG ATG TTC AGC GAG ATC ACT TTCCAA TCA GAT 1585 Phe Ala Asp Thr Trp Met Met Phe Ser Glu Ile Thr Phe GlnSer Asp 355 360 365 GCT GCA ATG TAT AAC AAC TCT GGA GCC CTT CCC ACC TCTCCT ATG GCA 1633 Ala Ala Met Tyr Asn Asn Ser Gly Ala Leu Pro Thr Ser ProMet Ala 370 375 380 CCC ACC ACC TAT GAT CCC ATG CTT AAA GTT GAT GAT AGCAAC ACT CGG 1681 Pro Thr Thr Tyr Asp Pro Met Leu Lys Val Asp Asp Ser AsnThr Arg 385 390 395 ATC CTG ATT GGT TGC TTG GTG GCC ATC ATC TTC ATC CTGCTG GCT ATC 1729 Ile Leu Ile Gly Cys Leu Val Ala Ile Ile Phe Ile Leu LeuAla Ile 400 405 410 415 ATC GTC ATC ATC CTG TGG AGG CAG TTC TGG CAG AAGATG CTA GAA AAG 1777 Ile Val Ile Ile Leu Trp Arg Gln Phe Trp Gln Lys MetLeu Glu Lys 420 425 430 GCT TCA CGG AGG ATG CTG GAT GAT GAA ATG ACA GTCAGC CTT TCC CTG 1825 Ala Ser Arg Arg Met Leu Asp Asp Glu Met Thr Val SerLeu Ser Leu 435 440 445 CCC AGC GAG TCC AGC ATG TTC AAT AAC AAC CGC TCCTCA TCA CCA AGT 1873 Pro Ser Glu Ser Ser Met Phe Asn Asn Asn Arg Ser SerSer Pro Ser 450 455 460 GAA CAG GAG TCC AAC TCT ACT TAT GAT CGA ATC TTCCCC CTT CGC CCT 1921 Glu Gln Glu Ser Asn Ser Thr Tyr Asp Arg Ile Phe ProLeu Arg Pro 465 470 475 GAC TAC CAG GAG CCA TCC AGA CTG ATC CGA AAG CTTCCA GAG TTT GCT 1969 Asp Tyr Gln Glu Pro Ser Arg Leu Ile Arg Lys Leu ProGlu Phe Ala 480 485 490 495 CCA GGA GAG GAG GAG TCA GGG TGC AGT GGT GTTGTG AAG CCG GCC CAG 2017 Pro Gly Glu Glu Glu Ser Gly Cys Ser Gly Val ValLys Pro Ala Gln 500 505 510 CCC AAT GGA CCT GAG GGC GTG CCC CAC TAT GCAGAA GCC GAC ATA GTG 2065 Pro Asn Gly Pro Glu Gly Val Pro His Tyr Ala GluAla Asp Ile Val 515 520 525 AAT CTC CAG GGA GTG ACA GGT GGC AAC ACC TACTGT GTG CCT GCT GTA 2113 Asn Leu Gln Gly Val Thr Gly Gly Asn Thr Tyr CysVal Pro Ala Val 530 535 540 ACC ATG GAT CTG CTA TCG GGG AAA GAT GTG GCTGTG GAA GAG TTC CCC 2161 Thr Met Asp Leu Leu Ser Gly Lys Asp Val Ala ValGlu Glu Phe Pro 545 550 555 AGG AAA CTG TTG GCC TTC AAG GAG AAG CTG GGAGAA GGC CAG TTT GGG 2209 Arg Lys Leu Leu Ala Phe Lys Glu Lys Leu Gly GluGly Gln Phe Gly 560 565 570 575 GAG GTT CAT CTC TGT GAA GTG GAG GGA ATGGAA AAA TTC AAA GAC AAA 2257 Glu Val His Leu Cys Glu Val Glu Gly Met GluLys Phe Lys Asp Lys 580 585 590 GAT TTT GCA CTA GAT GTC AGT GCC AAC CAGCCT GTC CTG GTG GCC GTG 2305 Asp Phe Ala Leu Asp Val Ser Ala Asn Gln ProVal Leu Val Ala Val 595 600 605 AAA ATG CTC CGA GCA GAT GCC AAC AAG AATGCC AGG AAT GAT TTT CTT 2353 Lys Met Leu Arg Ala Asp Ala Asn Lys Asn AlaArg Asn Asp Phe Leu 610 615 620 AAG GAG ATC AAG ATC ATG TCT CGG CTC AAGGAC CCA AAC ATC ATC CGT 2401 Lys Glu Ile Lys Ile Met Ser Arg Leu Lys AspPro Asn Ile Ile Arg 625 630 635 CTC TTA GCT GTG TGC ATC ACT GAG GAC CCGCTC TGC ATG ATC ACG GAA 2449 Leu Leu Ala Val Cys Ile Thr Glu Asp Pro LeuCys Met Ile Thr Glu 640 645 650 655 TAC ATG GAG AAT GGA GAT CTT AAT CAGTTT CTT TCT CGC CAC GAG CCT 2497 Tyr Met Glu Asn Gly Asp Leu Asn Gln PheLeu Ser Arg His Glu Pro 660 665 670 CTG AGT TCC TGT TCT AGT GAT GCC ACAGTC AGT TAC GCC AAC CTG AAG 2545 Leu Ser Ser Cys Ser Ser Asp Ala Thr ValSer Tyr Ala Asn Leu Lys 675 680 685 TTT ATG GCA ACC CAG ATT GCC TCT GGTATG AAG TAC CTT TCG TCT CTC 2593 Phe Met Ala Thr Gln Ile Ala Ser Gly MetLys Tyr Leu Ser Ser Leu 690 695 700 AAC TTT GTC CAC CGA GAT CTG GCC ACACGA AAC TGT TTA GTG GGC AAG 2641 Asn Phe Val His Arg Asp Leu Ala Thr ArgAsn Cys Leu Val Gly Lys 705 710 715 AAT TAC ACC ATC AAG ATA GCT GAT TTTGGC ATG AGC AGA AAC CTG TAC 2689 Asn Tyr Thr Ile Lys Ile Ala Asp Phe GlyMet Ser Arg Asn Leu Tyr 720 725 730 735 AGT GGT GAT TAC TAC CGG ATC CAGGGC CGG GCG GTG CTC CCC ATT CGC 2737 Ser Gly Asp Tyr Tyr Arg Ile Gln GlyArg Ala Val Leu Pro Ile Arg 740 745 750 TGG ATG TCC TGG GAA AGC ATC TTGCTG GGC AAA TTC ACC ACG GCA AGT 2785 Trp Met Ser Trp Glu Ser Ile Leu LeuGly Lys Phe Thr Thr Ala Ser 755 760 765 GAT GTG TGG GCC TTT GGG GTG ACTCTG TGG GAG ACC TTC ACC TTT TGC 2833 Asp Val Trp Ala Phe Gly Val Thr LeuTrp Glu Thr Phe Thr Phe Cys 770 775 780 CAG GAG CAG CCC TAT TCC CAG CTGTCG GAT GAG CAG GTT ATC GAG AAC 2881 Gln Glu Gln Pro Tyr Ser Gln Leu SerAsp Glu Gln Val Ile Glu Asn 785 790 795 ACT GGA GAG TTC TTC CGA GAC CAAGGG AGG CAG ATC TAT CTC CCT CAA 2929 Thr Gly Glu Phe Phe Arg Asp Gln GlyArg Gln Ile Tyr Leu Pro Gln 800 805 810 815 CCA GCC CTT TGC CCC GAC TCTGTG TAT AAG CTG ATG CTC AGC TGC TGG 2977 Pro Ala Leu Cys Pro Asp Ser ValTyr Lys Leu Met Leu Ser Cys Trp 820 825 830 AGA AGA GAA ACC AAG CAC CGGCCA TCC TTC CAG GAA ATA CAC CTC CTG 3025 Arg Arg Glu Thr Lys His Arg ProSer Phe Gln Glu Ile His Leu Leu 835 840 845 CTT CTT CAG CAA GGA GCC GAGT GATGATGCAT CAGCACCTGG CAGTGTTCCT 3077 Leu Leu Gln Gln Gly Ala Glu 850GTGGCCCAGA TCCTTCCCAC AAGACCTACT GCTCACCCAC ATC 3120 854 amino acidsamino acid linear protein 20 Met Ile Pro Ile Pro Arg Met Pro Leu Val LeuLeu Leu Leu Leu Leu 1 5 10 15 Ile Leu Gly Ser Ala Lys Ala Gln Val AsnPro Ala Ile Cys Arg Tyr 20 25 30 Pro Leu Gly Met Ser Gly Gly His Ile ProAsp Glu Asp Ile Thr Ala 35 40 45 Ser Ser Gln Trp Ser Glu Ser Thr Ala AlaLys Tyr Gly Arg Leu Asp 50 55 60 Ser Glu Glu Gly Asp Gly Ala Trp Cys ProGlu Ile Pro Val Gln Pro 65 70 75 80 Asp Asp Leu Lys Glu Phe Leu Gln IleAsp Leu Arg Thr Leu His Phe 85 90 95 Ile Thr Leu Val Gly Thr Gln Gly ArgHis Ala Gly Gly His Gly Ile 100 105 110 Glu Phe Ala Pro Met Tyr Lys IleAsn Tyr Ser Arg Asp Gly Ser Arg 115 120 125 Trp Ile Ser Trp Arg Asn ArgHis Gly Lys Gln Val Leu Asp Gly Asn 130 135 140 Ser Asn Pro Tyr Asp ValPhe Leu Lys Asp Leu Glu Pro Pro Ile Val 145 150 155 160 Ala Arg Phe ValArg Leu Ile Pro Val Thr Asp His Ser Met Asn Val 165 170 175 Cys Met ArgVal Glu Leu Tyr Gly Cys Val Trp Leu Asp Gly Leu Val 180 185 190 Ser TyrAsn Ala Pro Ala Gly Gln Gln Phe Val Leu Pro Gly Gly Ser 195 200 205 IleIle Tyr Leu Asn Asp Ser Val Tyr Asp Gly Ala Val Gly Tyr Ser 210 215 220Met Thr Glu Gly Leu Gly Gln Leu Thr Asp Gly Val Ser Gly Leu Asp 225 230235 240 Asp Phe Thr Gln Thr His Glu Tyr His Val Trp Pro Gly Tyr Asp Tyr245 250 255 Val Gly Trp Arg Asn Glu Ser Ala Thr Asn Gly Phe Ile Glu IleMet 260 265 270 Phe Glu Phe Asp Arg Ile Arg Asn Phe Thr Thr Met Lys ValHis Cys 275 280 285 Asn Asn Met Phe Ala Lys Gly Val Lys Ile Phe Lys GluVal Gln Cys 290 295 300 Tyr Phe Arg Ser Glu Ala Ser Glu Trp Glu Pro ThrAla Val Tyr Phe 305 310 315 320 Pro Leu Val Leu Asp Asp Val Asn Pro SerAla Arg Phe Val Thr Val 325 330 335 Pro Leu His His Arg Met Ala Ser AlaIle Lys Cys Gln Tyr His Phe 340 345 350 Ala Asp Thr Trp Met Met Phe SerGlu Ile Thr Phe Gln Ser Asp Ala 355 360 365 Ala Met Tyr Asn Asn Ser GlyAla Leu Pro Thr Ser Pro Met Ala Pro 370 375 380 Thr Thr Tyr Asp Pro MetLeu Lys Val Asp Asp Ser Asn Thr Arg Ile 385 390 395 400 Leu Ile Gly CysLeu Val Ala Ile Ile Phe Ile Leu Leu Ala Ile Ile 405 410 415 Val Ile IleLeu Trp Arg Gln Phe Trp Gln Lys Met Leu Glu Lys Ala 420 425 430 Ser ArgArg Met Leu Asp Asp Glu Met Thr Val Ser Leu Ser Leu Pro 435 440 445 SerGlu Ser Ser Met Phe Asn Asn Asn Arg Ser Ser Ser Pro Ser Glu 450 455 460Gln Glu Ser Asn Ser Thr Tyr Asp Arg Ile Phe Pro Leu Arg Pro Asp 465 470475 480 Tyr Gln Glu Pro Ser Arg Leu Ile Arg Lys Leu Pro Glu Phe Ala Pro485 490 495 Gly Glu Glu Glu Ser Gly Cys Ser Gly Val Val Lys Pro Ala GlnPro 500 505 510 Asn Gly Pro Glu Gly Val Pro His Tyr Ala Glu Ala Asp IleVal Asn 515 520 525 Leu Gln Gly Val Thr Gly Gly Asn Thr Tyr Cys Val ProAla Val Thr 530 535 540 Met Asp Leu Leu Ser Gly Lys Asp Val Ala Val GluGlu Phe Pro Arg 545 550 555 560 Lys Leu Leu Ala Phe Lys Glu Lys Leu GlyGlu Gly Gln Phe Gly Glu 565 570 575 Val His Leu Cys Glu Val Glu Gly MetGlu Lys Phe Lys Asp Lys Asp 580 585 590 Phe Ala Leu Asp Val Ser Ala AsnGln Pro Val Leu Val Ala Val Lys 595 600 605 Met Leu Arg Ala Asp Ala AsnLys Asn Ala Arg Asn Asp Phe Leu Lys 610 615 620 Glu Ile Lys Ile Met SerArg Leu Lys Asp Pro Asn Ile Ile Arg Leu 625 630 635 640 Leu Ala Val CysIle Thr Glu Asp Pro Leu Cys Met Ile Thr Glu Tyr 645 650 655 Met Glu AsnGly Asp Leu Asn Gln Phe Leu Ser Arg His Glu Pro Leu 660 665 670 Ser SerCys Ser Ser Asp Ala Thr Val Ser Tyr Ala Asn Leu Lys Phe 675 680 685 MetAla Thr Gln Ile Ala Ser Gly Met Lys Tyr Leu Ser Ser Leu Asn 690 695 700Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Lys Asn 705 710715 720 Tyr Thr Ile Lys Ile Ala Asp Phe Gly Met Ser Arg Asn Leu Tyr Ser725 730 735 Gly Asp Tyr Tyr Arg Ile Gln Gly Arg Ala Val Leu Pro Ile ArgTrp 740 745 750 Met Ser Trp Glu Ser Ile Leu Leu Gly Lys Phe Thr Thr AlaSer Asp 755 760 765 Val Trp Ala Phe Gly Val Thr Leu Trp Glu Thr Phe ThrPhe Cys Gln 770 775 780 Glu Gln Pro Tyr Ser Gln Leu Ser Asp Glu Gln ValIle Glu Asn Thr 785 790 795 800 Gly Glu Phe Phe Arg Asp Gln Gly Arg GlnIle Tyr Leu Pro Gln Pro 805 810 815 Ala Leu Cys Pro Asp Ser Val Tyr LysLeu Met Leu Ser Cys Trp Arg 820 825 830 Arg Glu Thr Lys His Arg Pro SerPhe Gln Glu Ile His Leu Leu Leu 835 840 845 Leu Gln Gln Gly Ala Glu 850171 base pairs nucleic acid single linear DNA Tyro-11 CDS 1..171 21 AACATC CTG GTC AAC AGT AAC CTG GTC TGC AAG GTG TCC GAC TTT GGC 48 Asn IleLeu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 CTCTCC AGA TTC CTG GAG GAG AAC TCC TCT GAT CCC ACC TAC ACA AGT 96 Leu SerArg Phe Leu Glu Glu Asn Ser Ser Asp Pro Thr Tyr Thr Ser 20 25 30 TCC CTGGGA GGA AAG ATT CCC ATC CGA TGG ACC GCC CCT GAG GCC ATT 144 Ser Leu GlyGly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 GCC TTC AGGAAA TTC ACG TCT GCC AGT 171 Ala Phe Arg Lys Phe Thr Ser Ala Ser 50 55 57amino acids amino acid linear protein 22 Asn Ile Leu Val Asn Ser Asn LeuVal Cys Lys Val Ser Asp Phe Gly 1 5 10 15 Leu Ser Arg Phe Leu Glu GluAsn Ser Ser Asp Pro Thr Tyr Thr Ser 20 25 30 Ser Leu Gly Gly Lys Ile ProIle Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 Ala Phe Arg Lys Phe Thr SerAla Ser 50 55 162 base pairs nucleic acid single linear DNA Tyro-12 CDS1..162 23 AAT TGC ATG TTG CGG GAT GAC ATG ACT GTC TGC GTG GCA GAC TTTGGC 48 Asn Cys Met Leu Arg Asp Asp Met Thr Val Cys Val Ala Asp Phe Gly 15 10 15 CTC TCT AAG AAG ATT TAC AGT GGT GAT TAT TAC CGC CAA GGC CGC ATT96 Leu Ser Lys Lys Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln Gly Arg Ile 20 2530 GCC AAA ATG CCT GTG AAG TGG ATC GCC ATA GAG AGC CTG GCG GAC CGA 144Ala Lys Met Pro Val Lys Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg 35 40 45GTC TAC ACA AGC AAG AGT 162 Val Tyr Thr Ser Lys Ser 50 54 amino acidsamino acid linear protein 24 Asn Cys Met Leu Arg Asp Asp Met Thr Val CysVal Ala Asp Phe Gly 1 5 10 15 Leu Ser Lys Lys Ile Tyr Ser Gly Asp TyrTyr Arg Gln Gly Arg Ile 20 25 30 Ala Lys Met Pro Val Lys Trp Ile Ala IleGlu Ser Leu Ala Asp Arg 35 40 45 Val Tyr Thr Ser Lys Ser 50 147 basepairs nucleic acid single linear DNA Tyro-13 CDS 1..147 25 AAT GTG CTGGTG TCT GAG GAC AAC GTG GCC AAA GTC AGT GAC TTT GGC 48 Asn Val Leu ValSer Glu Asp Asn Val Ala Lys Val Ser Asp Phe Gly 1 5 10 15 CTC ACT AAGGAA GCT TCC AGC ACT CAG GAC ACA GGC AAA CTG CCA GTC 96 Leu Thr Lys GluAla Ser Ser Thr Gln Asp Thr Gly Lys Leu Pro Val 20 25 30 AAG TGG ACA GCTCCT GAA GCC TTG AGA GAG AAG AAA TTT TCC ACC AAG 144 Lys Trp Thr Ala ProGlu Ala Leu Arg Glu Lys Lys Phe Ser Thr Lys 35 40 45 TCT 147 Ser 49amino acids amino acid linear protein 26 Asn Val Leu Val Ser Glu Asp AsnVal Ala Lys Val Ser Asp Phe Gly 1 5 10 15 Leu Thr Lys Glu Ala Ser SerThr Gln Asp Thr Gly Lys Leu Pro Val 20 25 30 Lys Trp Thr Ala Pro Glu AlaLeu Arg Glu Lys Lys Phe Ser Thr Lys 35 40 45 Ser 7 amino acids aminoacid Not Relevant linear protein 27 His Arg Asp Leu Ala Ala Arg 1 5 7amino acids amino acid Not Relevant linear protein 28 Asp Val Trp SerXaa Gly Xaa 1 5 8 amino acids amino acid Not Relevant linear protein 29Pro Xaa Xaa Trp Xaa Ala Pro Glu 1 5 68 amino acids amino acid NotRelevant linear protein 30 His Arg Asp Leu Ala Ala Arg Asn Val Leu ValLys Thr Pro Gln His 1 5 10 15 Val Lys Ile Thr Asp Phe Gly Leu Ala AspLeu Leu Gly Ala Glu Glu 20 25 30 Lys Glu Tyr His Ala Glu Gly Gly Lys ValPro Ile Lys Trp Met Ala 35 40 45 Leu Glu Ser Ile Leu His Arg Ile Tyr ThrHis Gln Ser Asp Val Trp 50 55 60 Ser Tyr Gly Val 65 68 amino acids aminoacid Not Relevant linear protein 31 His Arg Asp Leu Ala Ala Arg Asn CysMet Val Ala His Asp Phe Thr 1 5 10 15 Val Lys Ile Gly Asp Phe Gly MetThr Arg Asp Ile Tyr Glu Thr Asp 20 25 30 Tyr Tyr Arg Lys Gly Gly Lys GlyLeu Leu Pro Val Arg Trp Met Ala 35 40 45 Pro Glu Ser Leu Lys Asp Gly ValPhe Thr Thr Ser Ser Asp Met Trp 50 55 60 Ser Phe Gly Val 65 68 aminoacids amino acid Not Relevant linear protein 32 His Arg Asp Leu Ala AlaArg Asn Val Leu Ile Cys Glu Gly Lys Leu 1 5 10 15 Val Lys Ile Cys AspPhe His Leu Ala Arg Asp Ile Met Arg Asp Ser 20 25 30 Asn Tyr Ile Ser LysGly Ser Thr Tyr Leu Pro Leu Lys Trp Met Ala 35 40 45 Pro Glu Ser Ile PheAsn Ser Leu Tyr Thr Thr Leu Ser Asp Val Trp 50 55 60 Ser Phe Gly Ile 6568 amino acids amino acid Not Relevant linear protein 33 His Arg Asp LeuAla Ala Arg Asn Val Leu Ile Cys Glu Gly Lys Leu 1 5 10 15 Val Lys IleCys Asp Phe Gly Leu Ala Arg Asp Ile Met Arg Asp Ser 20 25 30 Asn Tyr IleIle Asp Gly Ser Thr Tyr Leu Pro Leu Lys Trp Met Ala 35 40 45 Pro Glu SerIle Phe Asn Ser Leu Tyr Thr Thr Leu Ser Asp Val Trp 50 55 60 Ser Phe GlyIle 65 43 amino acids amino acid Not Relevant linear protein 34 His ArgAsp Leu Ala Ala Arg Asn Val Leu Val Val Lys Ile Asp Phe 1 5 10 15 GlyLeu Ala Arg Asp Ile Tyr Gly Leu Pro Lys Trp Met Ala Pro Glu 20 25 30 SerTyr Thr Ser Asp Val Trp Ser Phe Gly Val 35 40 27 base pairs nucleic acidNot Relevant linear protein 35 GGAATTCCAT CGNGATTTNG CNGCNCG 27 55 aminoacids amino acid Not Relevant linear protein 36 Asn Cys Leu Val Gly GluAsn Ile Ile Leu Val Lys Val Ala Asp Phe 1 5 10 15 Gly Leu Ser Arg LeuMet Thr Gly Asp Thr Tyr Thr Ala Ile Ile Ala 20 25 30 Gly Ala Lys Phe ProIle Lys Trp Thr Ala Pro Glu Ser Leu Ala Tyr 35 40 45 Asn Lys Phe Ser IleLys Ser 50 55 55 amino acids amino acid Not Relevant linear protein 37Asn Cys Leu Val Gly Glu Asn Ile Ile Val Val Lys Val Ala Asp Phe 1 5 1015 Gly Leu Ser Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Ile Ile Ala 20 2530 Gly Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ser Leu Ala Tyr 35 4045 Asn Thr Pro Ser Ile Lys Ser 50 55 54 amino acids amino acid NotRelevant linear protein 38 Asn Cys Leu Val Thr Glu Lys Asn Val Leu LysIle Ser Asp Phe Gly 1 5 10 15 His Ser Arg Glu Glu Ala Asp Gly Val TyrAla Ala Ser Gly Gly Leu 20 25 30 Arg Gln Val Pro Val Lys Trp Thr Ala ProGlu Ala Leu Asn Tyr Gly 35 40 45 Arg Tyr Ser Ser Glu Ser 50 53 aminoacids amino acid Not Relevant linear protein 39 Asn Cys Leu Val Gly GluAsn Asn Thr Leu Lys Ile Ser Asp Phe Gly 1 5 10 15 Met Ser Arg Gln GluAsp Gly Gly Val Tyr Ser Ser Ser Gly Leu Lys 20 25 30 Gln Ile Pro Ile LysTrp Thr Ala Pro Glu Ala Leu His Tyr Gly Arg 35 40 45 Tyr Ser Ser Glu Ser50 53 amino acids amino acid Not Relevant linear protein 40 Asn Cys LeuVal Gly Ser Glu Asn Val Val Lys Val Ala Asp Phe Gly 1 5 10 15 Leu AlaArg Tyr Val Leu Asp Asp Gln Tyr Thr Ser Ser Gly Gly Thr 20 25 30 Lys PhePro Ile Lys Trp Ala Pro Pro Glu Val Leu Asn Tyr Thr Arg 35 40 45 Phe SerSer Lys Ser 50 54 amino acids amino acid Not Relevant linear protein 41Asn Ile Leu Val Asn Gln Asn Leu Cys Cys Lys Val Ser Asp Phe Gly 1 5 1015 Leu Thr Arg Leu Leu Asp Asp Phe Asp Gly Thr Tyr Glu Thr Gln Gly 20 2530 Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Leu Ala His Arg 35 4045 Ile Phe Thr Thr Ala Ser 50 55 amino acids amino acid Not Relevantlinear protein 42 Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val SerAsp Phe Gly 1 5 10 15 Leu Ser Arg Val Leu Glu Asp Asp Pro Glu Ala ThrTyr Thr Thr Ser 20 25 30 Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro GluAla Ile Ser Tyr 35 40 45 Arg Lys Phe Thr Ser Ala Ser 50 55 57 aminoacids amino acid Not Relevant linear protein 43 Asn Ile Leu Val Asn SerAsn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 Leu Ser Arg Tyr LeuGln Asp Asp Thr Ser Asp Pro Thr Tyr Thr Ser 20 25 30 Ser Leu Gly Gly LysIle Pro Val Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 Ala Tyr Arg Lys PheThr Ser Ala Ser 50 55 54 amino acids amino acid Not Relevant linearprotein 44 Asn Val Leu Val Lys Thr Pro Gln His Val Lys Ile Thr Asp PheGly 1 5 10 15 Leu Ala Lys Leu Leu Gly Ala Glu Glu Lys Glu Tyr His AlaGlu Gly 20 25 30 Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile LeuHis Arg 35 40 45 Ile Tyr Thr His Gln Ser 50 54 amino acids amino acidNot Relevant linear protein 45 Asn Val Leu Val Lys Ser Pro Asn His ValLys Ile Thr Asp Phe Gly 1 5 10 15 Leu Ala Arg Leu Leu Asp Ile Asp GluThr Glu Tyr His Ala Asp Gly 20 25 30 Gly Lys Val Pro Ile Lys Trp Met AlaLeu Glu Ser Ile Leu Arg Arg 35 40 45 Arg Phe Thr His Gln Ser 50 54 aminoacids amino acid Not Relevant linear protein 46 Asn Val Leu Val Thr GluAsp Asn Val Met Lys Ile Ala Asp Phe Gly 1 5 10 15 Leu Ala Arg Asp IleHis His Ile Asp Tyr Tyr Lys Lys Thr Thr Asn 20 25 30 Gly Arg Leu Pro ValLys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg 35 40 45 Ile Tyr Thr His GlnSer 50 54 amino acids amino acid Not Relevant linear protein 47 Asn ValLeu Val Thr Glu Asn Asn Val Met Lys Ile Ala Asp Phe Gly 1 5 10 15 LeuAla Arg Asp Ile Asn Asn Ile Asp Tyr Tyr Lys Lys Thr Thr Asn 20 25 30 GlyArg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg 35 40 45 ValTyr Thr His Gln Ser 50 54 amino acids amino acid Not Relevant linearprotein 48 Asn Val Leu Leu Ala Gln Gly Lys Ile Val Lys Ile Cys Asp PheGly 1 5 10 15 Leu Ala Arg Asp Ile Met His Asp Ser Asn Thr Val Ser LysGly Ser 20 25 30 Thr Phe Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile PheAsp Asn 35 40 45 Leu Thr Tyr Tyr Leu Ser 50 54 amino acids amino acidNot Relevant linear protein 49 Asn Met Leu Ile Cys Glu Gly Lys Leu ValLys Ile Cys Asp Phe Gly 1 5 10 15 Leu Ala Arg Asp Ile Met Arg Asp SerAsn Tyr Ile Ser Lys Gly Ser 20 25 30 Thr Phe Leu Pro Leu Lys Trp Met AlaPro Glu Ser Ile Phe Asn Ser 35 40 45 Leu Tyr Thr Thr Leu Ser 50 54 aminoacids amino acid Not Relevant linear protein 50 Asn Val Leu Leu Thr SerGly His Val Ala Lys Ile Gly Asp Phe Gly 1 5 10 15 Leu Ala Arg Asp IleMet Asn Asp Ser Asn Tyr Val Val Lys Gly Asn 20 25 30 Ala Arg Leu Pro ValLys Trp Met Ala Pro Glu Ser Ile Phe Asp Cys 35 40 45 Val Tyr Thr Tyr GlnSer 50 54 amino acids amino acid Not Relevant linear protein 51 Asn IleLeu Leu Ser Glu Asn Asn Val Val Lys Ile Cys Asp Phe Gly 1 5 10 15 LeuAla Arg Asp Ile Tyr Lys Asn Pro Asp Tyr Val Arg Arg Gly Asp 20 25 30 ThrArg Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Lys 35 40 45 ValTyr Ser Thr Lys Ser 50 54 amino acids amino acid Not Relevant linearprotein 52 Asn Cys Leu Val Gly Gln Gly Leu Val Val Lys Ile Gly Asp PheGly 1 5 10 15 Met Ser Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val GlyGly Arg 20 25 30 Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile LeuTyr Arg 35 40 45 Lys Phe Thr Thr Glu Ser 50 54 amino acids amino acidNot Relevant linear protein 53 Asn Cys Leu Val Gly Glu Asn Leu Leu ValLys Ile Gly Asp Phe Gly 1 5 10 15 Met Ser Arg Asp Val Tyr Ser Thr AspTyr Tyr Arg Val Gly Gly Arg 20 25 30 Thr Met Leu Pro Ile Arg Trp Met ProPro Glu Ser Ile Met Tyr Arg 35 40 45 Lys Phe Thr Thr Glu Ser 50 54 aminoacids amino acid Not Relevant linear protein 54 Asn Cys Met Val Ala GluAsp Phe Thr Val Lys Ile Gly Asp Phe Gly 1 5 10 15 Met Thr Arg Asp IleTyr Glu Thr Asp Tyr Tyr Arg Lys Gly Gly Lys 20 25 30 Gly Leu Leu Pro ValArg Trp Met Ser Pro Glu Ser Leu Lys Asp Gly 35 40 45 Val Phe Thr Thr HisSer 50

1. Substantially pure protein(s), or functional fragments thereof,characterized as having a tyrosine kinase domain and a tissue expressionpattern characteristic of at least one receptor protein-tyrosine kinasesubtype selected from the group consisting of tyro-1, tyro-2, tyro-4,tyro-5, tyro-6, tyro-7, tyro-8, tyro-10, tyro-11, and tyro-12.